Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
1
DESCRIPTION
NUCLEOZYMES WITH ENDONUCLEASE ACTIVITY
Background of the Invention
This patent application claims priority from Breaker et al., USSN
(60/193,646), filed March
31, 2000, entitled "NUCLEOZYMES WITH ENDONUCLEASE ACTIVITY" and from Breaker
et
al., USSN (60/181,360), filed February 8, 2000, entitled "NUCLEIC ACID
CATALYSTS WITH
ENDONUCLEASE ACTIVITY". These patent applications are hereby incorporated by
reference
herein in their entirety including the drawings.
This invention relates to nucleic acid molecules (nucleozymes), including
deoxyribonucleic
acid molecules, with catalytic activity and derivatives thereof.
The following is a brief description of enzymatic nucleic acid molecules. This
summary is not
meant to be complete but is provided only for understanding of the invention
that follows. This
summary is not an admission that all of the work described below is prior art
to the claimed
invention.
Enzymatic nucleic acid molecules are nucleic acid molecules capable of
catalyzing one or
more of a variety of reactions, including the ability to repeatedly cleave
(multiple turnover) other
separate nucleic acid molecules in a nucleotide base sequence-specific manner.
Such enzymatic
nucleic acid molecules can be used, for example, to target virtually any RNA
transcript (Zaug et al.,
324, Nature 429 1986; Cech, 260 JAMA 3030, 1988).
Because of their sequence-specificity, tans-cleaving enzymatic nucleic acid
molecules show
promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann.
Rep. Med.
Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037).
Enzymatic
nucleic acid molecules can be designed to cleave specific RNA targets within
the background of
cellular RNA. Such a cleavage event renders the mRNA non-functional and
abrogates protein
expression from that RNA. In this manner, synthesis of a protein associated
with a disease state can
be selectively inhibited.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
2
In general, enzymatic nucleic acids with RNA cleaving activity act by first
binding to a target
RNA. Such binding occurs through the target-binding portion of a enzymatic
nucleic acid which is
held in close proximity to an enzymatic portion of the molecule that acts to
cleave the target RNA.
Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA
through
complementary base pairing, and once bound to the correct site, acts
enzymatically to cut the target
RNA. Strategic cleavage of such a target RNA will destroy its ability to
direct synthesis of an
encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA
target, it is
released from that RNA to search for another target and can repeatedly bind
and cleave new targets.
Several approaches have been used to evolve new nucleic acid catalysts, for
example, in vitro
selection and/or in vitro evolution strategies (Orgel, 1979, Proc. R. Soc.
London, B 205, 435). These
approaches have been used to evolve new nucleic acid catalysts capable of
catalyzing a variety of
reactions, such as cleavage and ligation of phosphodiester linkages and amide
linkages, (Joyce,
1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce,
1992, Scientific American
267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et a1.,1993, Science
261:1411-1418;
Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB .L, 9, 1183; Breaker,
1996, Curr. Op.
Biotech., 7, 442).
The development of ribozymes that are optimal for catalytic activity would
contribute
significantly to any strategy that employs RNA-cleaving ribozymes for the
purpose of regulating
gene expression. The hammerhead ribozyme, for example, functions with a
catalytic rate (kcat) of ~l
min-1 in the presence of saturating (10 mM) concentrations of Mg2+ cofactor.
However, the rate for
this ribozyme in Mg2+ concentrations that are closer to those found inside
cells (0.5 - 2 mM) can be
10- to 100-fold slower. In contrast, the RNase P holoenzyme can catalyze pre-
tRNA cleavage with a
kcat of ~30 min-1 under optimal assay conditions. An artificial 'RNA ligase'
ribozyme has been
shown to catalyze the corresponding self modification reaction with a rate of
100 min-1. In
addition, it is known that certain modified hammerhead ribozymes that have
substrate binding arms
made of DNA catalyze RNA cleavage with multiple turn-over rates that approach
100 min-1.
Finally, replacement of a specific residue within the catalytic core of the
hammerhead with certain
nucleotide analogues gives modified ribozymes that show as much as a 10-fold
improvement in
catalytic rate. These findings demonstrate that ribozymes can promote chemical
transformations
with catalytic rates that are significantly greater than those displayed in
vitro by most natural self
cleaving ribozymes. It is then possible that the structures of certain self
cleaving ribozymes may be
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
3
optimized to give maximal catalytic activity, or that entirely new RNA motifs
can be made that
display significantly faster rates for RNA phosphodiester cleavage.
An extensive array of site-directed mutagenesis studies have been conducted
with ribozymes
such as the hammerhead ribozyme and the hairpin ribozyme to probe
relationships between
nucleotide sequence and catalytic activity. These systematic studies have made
clear that most
nucleotides in the conserved core of the ribozyme cannot be mutated without
significant loss of
catalytic activity. In contrast, a combinatorial strategy that simultaneously
screens a large pool of
mutagenized ribozymes for RNAs that retain catalytic activity could be used
more efficiently to
define immutable sequences and to identify new ribozyme variants.
Tang et al., 1997, RNA 3, 914, reported novel ribozyme sequences with
endonuclease activity,
where the authors used an in vitro selection approach to isolate these
ribozymes.
Vaish et al., 1998 PNAS 95, 2158-2162, describes the in vitro selection of a
hammerhead-like
ribozyme with an extended range of cleavable triplets.
Breaker, International PCT publication No. WO 98/43993, describes the in vitro
selection of
hammerhead-like ribozymes with sequence variants encompassing the catalytic
core.
Catalytic nucleic acid molecules until recently were all known to require the
presence of 2'-
hydroxyl (2'-OH) groups within the molecule for their enzymatic function.
Usman and co-workers
recently reported that nucleic acid molecules lacking a 2'-hydroxyl group,
such as DNA molecules,
can catalyze a chemical reaction (Chartrand et al., 1995, Nucleic Acids
Research, 23(20), 4092-
4096; Usman et al., US Patent No. 5,861,288). There have also been several
reports of non-2'-OH
containing nucleic acid molecules that are capable of catalyzing chemical
reactions (Li and Breaker,
1999, Cur. Opin. Struct. Biol., 9, 315-323; Breaker, 1999, Nature
Biotechnology, 17, 422-423). The
use of in vitro selection and/or in vitro evolution techniques applied to
random pools of single-
stranded DNA has provided catalytic DNA, or deoxyribozymes, capable of
catalyzing the cleavage
of RNA (Usman et al., US Patent No. 5,861,288; Breaker and Joyce, 1994, Chem.
Biol., 1, 223-229;
Breaker and Joyce, 1995, Chem. Biol., 655-660), facilitation of the ligation
of chemically activated
DNA (Cuenoud and Szostak, 1995, Nature, 375, 611-614), and the metallation of
porphyrin rings
(Li and Sen, 1996, Nat. Struct. Biol., 3, 743-747).
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
4
The cleavage of RNA by transesterification via catalytic DNA structures is
often dependant on
metal ion cofactors, such as Mg2+, Mn2+, Zn2+, pb2+, Ca2+, and Cd2+, or an
amino acid cofactor
such as histidine (Roth and Breaker, 1998, PNAS USA., 95, 6027-6031). However,
the native
structure of DNA alone may be sufficient in providing the chemical groups that
are responsible for
catalysis (Geyer and Sen, 1997, Chem. Biol., 4, 579-593).
Joyce and Breaker, US Patent No. 5,807,718, describe specific magnesium
dependent RNA
cleaving DNA enzymes with defined structural and target sequence constraints.
Summary of the Invention
This invention relates to nucleic acid molecules with catalytic activity,
which are particularly
useful for cleavage of RNA or DNA. The nucleic acid catalysts of the instant
invention are distinct
from other nucleic acid catalysts known in the art. The nucleic acid catalysts
of the instant invention
do not share sequence homology with other known enzymatic nucleic acid
molecules, such as other
known DNA enzymes. Specifically, the nucleic acid catalysts of the instant
invention are capable of
catalyzing an intermolecular or intramolecular endonuclease reaction.
In a preferred embodiment, the invention features a nucleic acid molecule with
catalytic
activity having either the formulae I or II:
Formula I
3' X Z Y 5'
Formula II
3' X W V R Y 5'
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
In the above formulae I and II, X and Y are independently oligonucleotides of
length sufficient
to stably interact (e.g., by forming hydrogen bonds with complementary
nucleotides in the target)
with a target nucleic acid molecule (the target can be an RNA, DNA or RNA/DNA
mixed polymers,
including polymers that may include base, sugar, and/or phosphate nucleotide
modifications; such
5 modifications are preferably naturally occurring modifications), preferably,
the length of X and Y
are independently between 3-20 nucleotides long, e.g., specifically, 3, 4, S,
6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 17, and 20); X and Y may have the same lengths or may have different
lengths;
represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage
or others known in the
art); Z is independently a nucleotide sequence comprising 5'-
GATGCAGCTGGGGAGGCGTTT-3'
(SEQ ID NO 103), R is independently a nucleotide sequence comprising 5'-GGGGA-
3', V
represents a nucleotide or non-nucleotide linker, which may be present or
absent, (i.e., the molecule
is assembled from two separate molecules), but when present, is a nucleotide
and/or non-nucleotide
linker, which may comprise a single-stranded and/or double-stranded region; W
is independently a
nucleotide sequence selected from the group comprising 5'-TGGGGAAGCACAGGGT-3'
(SEQ ID
NO 104), 5'-TGGGGAAGCTCTGGGT-3' (SEQ ID NO 105), 5'-TGGGGAAGCACAGGGT-3'
(SEQ ID NO 106), and 5'-TGGGGAAGCACATGGT-3' (SEQ ID NO 107); additions,
deletions,
and substitutions to these sequences may be made without significantly
altering the activity of the
molecules and are hence within the scope of the invention; and C, G, A, and T
represent cytidine,
guanosine, adenosine and thymidine nucleotides, respectively. The nucleotides
in each of the
formulae I and II are unmodified or modified at the sugax, base, and/or
phosphate as known in the
art.
In a preferred embodiment, the nucleotide linker (V) is a nucleic acid
sequence selected from
the group consisting of 5'-ACCTGAGGG-3', 5'-GCGTTAG-3' and 5'-
AGGAAGCATCTTATGCGACC-3' (SEQ ID NO 108).
The nucleotide linker V is preferably 5-40 nucleotides in length, more
preferably 7-20
nucleotides in length and still more preferably 7-12 nucleotides in length.
In yet another embodiment, the nucleotide linker (V) is a nucleic acid
aptamer, such as an
ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a
review, see Gold
et al., 1995, Annu. Rev. Biochem., 64, 763; and Szostak & Ellington, 1993, in
The RNA World, ed.
Gesteland and Atkins, pp. 511, CSH Laboratory Press). A "nucleic acid aptamer"
as used herein is
meant to indicate a nucleic acid sequence capable of interacting with a
ligand. The ligand can be
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
6
any natural or synthetic molecule, including but not limited to a resin,
metabolites, nucleosides,
nucleotides, drugs, toxins, transition state analogs, peptides, lipids,
proteins, amino acids, nucleic
acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria
and others.
In another embodiment, the non-nucleotide linker (V) is as defined herein. The
term "non-
nucleotide" as used herein includes either abasic nucleotide, polyether,
polyamine, polyamide,
peptide, carbohydrate, lipid, or polyhydrocarbon compounds. These compounds
can be incorporated
into a nucleic acid chain in the place of one or more nucleotide units,
including either sugar and/or
phosphate substitutions, and allows the remaining bases to exhibit their
enzymatic activity. The
group or compound is abasic in that it does not contain a commonly recognized
nucleotide base,
such as adenine, guanine, cytosine, uracil or thymine. The terms "abasic" or
"abasic nucleotide" as
used herein encompass sugar moieties lacking a base or having other chemical
groups in place of a
nucleotide base at the 1' position. Specific examples of non-nucleotides
include those described by
Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res.
1987, 15:3113; Cload
and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J.
Am. Chem. Soc.
1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry
1993, 32:1751;
Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &
Nucleotides 1991,
10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al.,
Biochemistry 1991, 30:9914;
Arnold et al., International Publication No. WO 89/02439; Usman et al.,
International Publication
No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and
Ferentz and
Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by
reference herein. Thus, in a
preferred embodiment, the invention features an enzymatic nucleic acid
molecule having one or
more non-nucleotide moieties, and having enzymatic activity to cleave an RNA
or DNA molecule.
By 'nucleozyme' or 'DNA enzyme' or 'DNAzyme' or "deoxyribozyme" as used herein
is
meant, an enzymatic nucleic acid molecule that does not require the presence
of a ribonucleotide (2'-
OH) group in the molecule for its activity. These molecules are also referred
to as catalytic DNA,
nucleic acid catalysts, restriction endonucleases, catalytic oligonucleotides,
and enzymatic DNA
molecules. In particular embodiments, the enzymatic nucleic acid molecule may
have an attached
linkers) or other attached or associated groups, moieties, or chains
containing one or more
nucleotides with 2'-OH groups. DNAzymes can be synthesized chemically or
expressed
endogenously in vivo, by means of a single stranded DNA vector or equivalent
thereof.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
7
By "enzymatic nucleic acid molecule" it is meant a nucleic acid molecule which
has
complementarity in a substrate binding region to a specified gene target, and
also has an enzymatic
activity which is active to specifically cleave target RNA. That is, the
enzymatic nucleic acid
molecule is able to intermolecularly cleave RNA and thereby inactivate a
target RNA molecule.
These complementary regions allow sufficient hybridization of the enzymatic
nucleic acid molecule
to the target RNA and thus permit cleavage. One hundred percent
complementarity is preferred, but
complementarity as low as 50-75% can also be useful in this invention (see for
example Werner and
Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999,
Antisense and
Nucleic Acid Drug Dev., 9, 25-31). The nucleic acids can be modified at the
base, sugar, and/or
phosphate groups.
The enzymatic nucleic acid molecule (e.g., the molecules of formulae I and II)
of the instant
invention are capable of catalyzing (altering the velocity and/or rate of) a
variety of reactions
including the ability to repeatedly cleave (multiple turnover) other separate
nucleic acid molecules
(endonuclease activity) in a nucleotide base sequence-specific manner. Such a
molecule with
endonuclease activity may have complementarity in a substrate binding region
(e.g. X and Y in
formulae I and II) to a specified gene target, and has an enzymatic activity
that specifically cleaves
RNA or DNA in that target. That is, the nucleic acid molecule with
endonuclease activity is able to
intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate
a target RNA or
DNA molecule. The complementarity functions to allow sufficient hybridization
of the enzymatic
DNA molecule to the target RNA or DNA to allow the cleavage to occur. 100%
complementarity is
preferred, but complementarity as low as 50-75% may also be useful in this
invention. The nucleic
acids can be modified at the base, sugar, and/or phosphate groups. All that is
required, as will be
readily recognized by persons skilled in the art, is that the enzymatic
nucleic acid molecule not be
dependent on the presence of a ribonucleotide in the molecule for its
catalytic activity.
In preferred embodiments, the enzymatic nucleic acids of formulae I and II
includes one or
more stretches of non-ribonucleotide containing oligonucleotide, which provide
the enzymatic
activity of the molecule. The necessary non-ribonucleotide components are
known in the art.
Thus, in one preferred embodiment, the invention features DNA enzymes that
inhibit gene
expression and/or cell proliferation. These chemically or enzymatically
synthesized nucleic acid
molecules contain substrate binding domains that bind to accessible regions of
specific target
nucleic acid molecules. The nucleic acid molecules also contain domains that
catalyze the cleavage
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
8
of target. Upon binding, the enzymatic nucleic acid molecules cleave the
target molecules,
preventing, for example, translation and protein accumulation. In the absence
of the expression of
the target gene, cell proliferation, for example, is inhibited. In another
aspect of the invention,
enzymatic nucleic acid molecules that cleave target molecules are expressed
from a single stranded
DNA intracellular expression vector. Preferably, the vectors capable of
expressing the DNA
enzymes are delivered as described below, and persist in target cells.
Suitable vectors can be used
that provide for transient expression of DNA enzymes. Such vectors can be
repeatedly administered
as necessary. Once expressed, the DNA enzymes cleave the target mRNA. Delivery
of DNA
enzyme expressing vectors can be systemic, such as by intravenous or
intramuscular administration,
by administration to target cells ex-planted from the patient followed by
reintroduction into the
patient, or by any other means that would allow for introduction into the
desired target cell (for a
review, see Couture and Stinchcomb, 1996, TIG., 12, 510).
In a preferred embodiment, the invention features a nucleic acid molecule with
catalytic
activity having either the formulae III and IV:
Formula III
~ (N)n U G D X 3'
(N)m A G A - (N)D (N)p Y 5
\/
Formula IV
3' X Z Y 5'
In the above formulae III and IV, each N represents independently a nucleotide
or a non-
nucleotide linker, which can be same or different; X and Y are independently
oligonucleotides of
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
9
length sufficient to stably interact (e.g., by forming hydrogen bonds with
complementary nucleotides
in the target) with a target nucleic acid molecule (the target can be an RNA,
DNA or RNA/DNA
mixed polymers, including polymers that can include base, sugar, and/or
phosphate nucleotide
modifications; such modifications are preferably naturally occurnng
modifications), preferably, the
length of X and Y are independently between 3-20 nucleotides long, e.g.,
specifically, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 17, and 20); X and Y can have the same lengths or
can have different
lengths; m, n, o, and p are integers independently greater than or equal to 1
and preferably less than
about 100, specifically 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50;
wherein if (I~m and (N)n
and/or (N)o and (N)p are nucleotides, (lam and (N)n and/or (loo and (I~p are
optionally able to
interact by hydrogen bond interaction; preferably, (N)m and (l~n and/or (loo
and (N)p
independently form 1, 2, 3, 4, 5, 6, 7, 8, 9 base-paired stem structures; D is
U, G or A; L~ and L2 are
independently linkers, which can be the same or different and which can be
present or absent (i.e.,
the molecule is assembled from two separate molecules), but when present, are
nucleotide and/or
non-nucleotide linkers, which can comprise a single-stranded and/or double-
stranded region;
represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage
or others known
in the art); ~ represents a base-pair interaction; Z is independently a
nucleotide sequence selected
from the group comprising 5'-AGAUAACGUGAAGAU-3' (SEQ ID NO 109) and 5'-
AAUGGCCUAUCGGUGCGA-3' (SEQ ID NO 110), additions, deletions, and substitutions
to these
sequences can be made without significantly altering the activity of the
molecules and are hence
within the scope of the invention; and C, G, A, and U represent cytidine,
guanosine, adenosine and
uridine nucleotides, respectively. The nucleotides in each of the formulae III
and IV are unmodified
or modified at the sugar, base, and/or phosphate as known in the art.
In a preferred embodiment, the invention features nucleic acid molecules of
Formula III,
where the sequence of oligonucleotide (N)m is selected from the group
consisting of 5'-AC-3', 5'-
GC-3', and 5'-CG-3'.
In another preferred embodiment, the invention features nucleic acid molecules
of Formula III,
where the sequence of oligonucleotide (N)n is selected from the group
consisting of 5'-GU-3', 5'-
GC-3', and. 5'-CG-3'.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
In yet another preferred embodiment, the invention features nucleic acid
molecules of Formula
III, where the sequence of oligonucleotide (N)o is selected from the group
consisting of 5'-AUUG-
3', 5'-UUG-3', 5'-UUC-3', and 5'-UAG-3'.
In an additional preferred embodiment, the 'nvention features nucleic acid
molecules of
5 Formula III, where the sequence of oligonucleotide (N)p is selected from the
group consisting of 5'
CAAU-3', 5'-CAA-3', 5'-GAA-3', and 5'-CUA-3'.
In another embodiment, the nucleotide linker (L~) is a nucleic acid sequence
selected from the
group consisting of 5'-CUUAA-3' and 5'-CUAAA-3'.
In another embodiment, the nucleotide linker (L2) is a nucleic acid sequence
selected from the
10 group consisting of 5'-UGUGAA-3' and 5'-GUGA-3'.
In yet another embodiment, the nucleotide linker (L~ and/or L2) is a nucleic
acid aptamer,
such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and
others (for a review
see Gold et al., 1995, Annu. Rev. Biochem., 64, 763; and Szostak & Ellington,
1993, in The RNA
World, ed. Gesteland and Atkins, pp. 511, CSH Laboratory Press). A "nucleic
acid aptamer" as used
herein is meant to indicate a nucleic acid sequence capable of interacting
with a ligand. The ligand
can be any natural or synthetic molecule, including but not limited to a
resin, metabolites,
nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides,
lipids, proteins, amino
acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells,
viruses, bacteria and others.
In another embodiment, the non-nucleotide linker (L~ and/or L2) is as defined
herein. The
term "non-nucleotide" as used herein include either abasic nucleotide,
polyether, polyamine,
polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds. These
compounds can be
incorporated into a nucleic acid chain in the place of one or more nucleotide
units, including either
sugar and/or phosphate substitutions, and allows the remaining bases to
exhibit their enzymatic
activity. The group or compound is abasic in that it does not contain a
commonly recognized
nucleotide base, such as adenine, guanine, cytosine, uracil or thymine. The
terms "abasic" or "abasic
nucleotide" as used herein encompass sugar moieties lacking a base or having
other chemical groups
in place of a nucleotide base at the 1' position. Specific examples of non-
nucleotides include those
described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic
Acids Res. 1987,
15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991,113:6324; Richardson and
Schepartz, J. Am.
Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and
Biochemistry 1993,
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
11
32:1751; Durand et al., Nucleic Acicls Res. 1990, 18:6353; McCurdy et al.,
Nucleosides &
Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono
et al., Biochemistry
1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman
et al.,
International Publication No. WO 95/06731; Dudycz et al., International
Publication No. WO
95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby
incorporated by
reference herein. Thus, in a preferred embodiment, the invention features an
enzymatic nucleic acid
molecule having one or more non-nucleotide moieties, and having .enzymatic
activity to cleave an
RNA or DNA molecule.
In preferred embodiments, the enzymatic nucleic acids of formulae III and IV
includes one or
more stretches of RNA, which provide the enzymatic activity of the molecule,
linked to the non-
nucleotide moiety. The necessary RNA components are known in the art (see for
e.g., Usman et al.,
supra).
Thus, in one preferred embodiment, the invention features ribozymes that
inhibit gene
expression and/or cell proliferation. These chemically or enzymatically
synthesized nucleic acid
molecules contain substrate binding domains that bind to accessible regions of
specific target
. nucleic acid molecules. The nucleic acid molecules also contain domains that
catalyze the cleavage
of target. Upon binding, the enzymatic nucleic acid molecules cleave the
target molecules,
preventing, for example, translation and protein accumulation. In the absence
of the expression of
the target gene, cell proliferation, for example, is inhibited. In another
aspect of the invention,
enzymatic nucleic acid molecules that cleave target molecules are expressed
from transcription units
inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA
plasmids or viral
vectors. Ribozyme expressing viral vectors could be constructed based on, but
not limited to, adeno-
associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the
recombinant vectors capable
of expressing the ribozymes are delivered as described below, and persist in
target cells.
Alternatively, viral vectors can be used that provide for transient expression
of ribozymes. Such
vectors can be repeatedly administered as necessary. Once expressed, the
ribozymes cleave the
target mRNA. Delivery of ribozyme expressing vectors can be systemic, such as
by intravenous or
intramuscular administration, by administration to target cells ex-planted
from the patient followed
by reintroduction into the patient, or by any other means that would allow for
introduction into the
desired target cell (for a review, see Couture and Stinchcomb, 1996, TIG., 12,
510).
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
12
"Complementarity" refers to the ability of a nucleic acid to form hydrogen
bonds) with
another RNA sequence by either traditional Watson-Crick or other non-
traditional types. In
reference to the nucleic molecules of the present invention, the binding free
energy for a nucleic acid
molecule with its target or complementary sequence is sufficient to allow the
relevant function of the
nucleic acid to proceed, e.g., cleavage via a DNAzyme. Determination of
binding free energies for
nucleic acid molecules is well known in the art (see, e.g., Turner et al.,
1987, CSH Symp. Quant.
Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-
9377; Turner et al.,
1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates
the percentage of
contiguous residues in a nucleic acid molecule which can form hydrogen bonds
(e.g., Watson-Crick
base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out
of 10 being 50%, 60%,
70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that
all the
contiguous residues of a nucleic acid sequence will hydrogen bond with the
same number of
contiguous residues in a second nucleic acid sequence.
By "sufficient length" is meant an oligonucleotide of greater than or equal to
3 nucleotides that
is of a length great enough to provide the intended function under the
expected condition. For
example, for binding arms of enzymatic nucleic acid "sufficient length" means
that the binding arm
sequence is long enough to provide stable binding to a target site under the
expected binding
conditions. Preferably, the binding arms are not so long as to prevent useful
turnover of the nucleic
acid molecule.
By "stably interact" is meant interaction of the oligonucleotides with target
nucleic acid (e.g.,
by forming hydrogen bonds with complementary nucleotides in the target under
physiological
conditions) that is sufficient to the intended purpose (e.g., cleavage of
target RNA by an enzyme).
By "inhibit" it is meant that the activity of a given protein or level of RNAs
or equivalent
RNAs encoding one or more protein subunits of a given protein target is
reduced below that
observed in the absence of the nucleic acid molecules of the invention. In one
embodiment,
inhibition with enzymatic nucleic acid molecule preferably is below that level
observed in the
presence of an enzymatically inactive or attenuated molecule that is able to
bind to the same site on
the target RNA, but is unable to cleave that RNA. In another embodiment,
inhibition of target genes
with the nucleic acid molecule of the instant invention is greater than in the
presence of the nucleic
acid molecule than in its absence.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
13
By "nucleic acid molecule" as used herein is meant a molecule having
nucleotides. The
nucleic acid can be single, double, or multiple stranded and may comprise
modified or unmodified
nucleotides or non-nucleotides or various mixtures and combinations thereof.
By "gene" it is meant a nucleic acid that encodes an RNA, for example, nucleic
acid sequences
including but not limited to structural genes encoding a polypeptide.
By "RNA" is meant a molecule comprising at least one ribonucleotide residue.
By
"ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2'
position of a beta-D-ribo-
furanose moiety.
As used in herein "cell" is used in its usual biological sense, and does not
refer to an entire
multicellular organism, e.g., specifically does not refer to a human. The cell
may be present in an
organism which may be a human but is preferably a non-human multicellular
organism, e.g., birds,
plants and mammals such as cows, sheep, apes, monkeys, swine, dogs, and cats.
The cell may be
prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant
cell).
By "patient" is meant an organism which is a donor or recipient of explanted
cells or the cells
themselves. "Patient" also refers to an organism to which enzymatic nucleic
acid molecules can be
administered. Preferably, a patient is a mammal or mammalian cells. More
preferably, a patient is a
human or human cells.
By "vectors" is meant any nucleic acid- and/or viral-based technique used to
deliver a desired
nucleic acid.
In another preferred embodiment, catalytic activity.of the molecules described
in the instant
invention can be optimized. Modifications which enhance their efficacy in
cells, and removal of
bases from stem loop structures to shorten enzymatic nucleic acid molecule
synthesis times and
reduce chemical requirements are desired. Catalytic activity of the molecules
described in the
instant invention can be optimized as described by Usman et al., US Patent No.
5,861,288. The
details will not be repeated here, but include altering the length of the
enzymatic nucleic acid
molecule binding arms, or chemically synthesizing enzymatic nucleic acid
molecules with
modifications (base, sugar and/or phosphate) that prevent their degradation by
serum nucleases
and/or enhance their enzymatic activity (see e.g., Eckstein et al.,
International Publication No. WO
92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science
253, 314; Usman and
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
14
Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al., International
Publication No. WO
93/1 S 187; and Rossi et al., International Publication No. WO 91/03162;
Sproat, US Patent No.
5,334,711; and Burgin et al., supra; all of these describe various chemical
modifications that can be
made to the base, phosphate and/or sugar moieties of enzymatic nucleic acid
molecules). All these
publications are hereby incorporated by reference herein.
By "enhanced enzymatic activity" is meant to include activity measured in
cells and/or in vivo
where the activity is a reflection of both catalytic activity and enzymatic
nucleic acid molecule
stability.
In yet another preferred embodiment, nucleic acid catalysts having chemical
modifications
which maintain or enhance enzymatic activity are provided. Such nucleic acid
is also generally
more resistant to nucleases than unmodified nucleic acid. Thus, in a cell
and/or in vivo the activity
may not be significantly lowered. As exemplified herein such enzymes are
useful in a cell and/or in
vivo even if activity over all is reduced 10-fold (Burgin et al., 1996,
Biochemistry, 35, 14090).
In a preferred embodiment, the invention provides a method for producing a
class of
enzymatic nucleic acid molecule-based gene inhibiting agents which exhibit a
high degree of
specificity for the RNA of a desired target. For example, the enzymatic
nucleic acid molecule is
preferably targeted to a highly conserved sequence region of target RNAs
encoding target proteins
such that specific treatment of a disease or condition can be provided with
either one or several
nucleic acid molecules of the invention. Such nucleic acid molecules can be
delivered exogenously
to specific tissue or cellular targets as required. Alternatively, the nucleic
acid molecules (e.g., DNA
enzymes) can be expressed from single stranded DNA expression vectors that are
delivered to
specific cells.
In a preferred embodiment, an expression vector comprising a nucleic acid
sequence encoding
at least one of the nucleic acid catalysts of the instant invention is
disclosed. The nucleic acid
sequence encoding the nucleic acid catalyst of the instant invention is
operable linked in a manner
which allows expression of that nucleic acid molecule.
In one embodiment, the expression vector comprises: a transcription initiation
region (e.g.,
eukaryotic pol I, II or III initiation region); b) a transcription termination
region (e.g., eukaryotic pol
I, II or III termination region); c) a nucleic acid sequence encoding at least
one of the nucleic acid
catalyst of the instant invention; and wherein said sequence is operably
linked to said initiation
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
region and said termination region, in a manner which allows expression and/or
delivery of said
nucleic acid molecule. The vector may optionally include'an open reading frame
(ORF) for a
protein operably linked on the S' side or the 3'-side of the sequence encoding
the nucleic acid
catalyst of the invention; and/or an intron (intervening sequences).
5 Another means of accumulating high concentrations of a ribozyme(s) within
cells is to
incorporate the ribozyme-encoding sequences into a DNA or RNA expression
vector. Transcription
of the ribozyme sequences are driven from a promoter for eukaryotic RNA
polymerase I (pol I),
RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from
pol II or pol III
promoters will be expressed at high levels in all cells; the levels of a given
pol II promoter in a given
10 cell type will depend on the nature of the gene regulatory sequences
(enhancers, silencers, etc.)
present nearby. Prokaryotic RNA polymerase promoters are also used, providing
that the
prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-
Stein and Moss,
1990 Proc. Natl. Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993 Nucleic
Acids Res., 21, 2867-
72; Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol.
Cell. Biol., 10, 4529-
15 37). Several investigators have demonstrated that ribozymes expressed from
such promoters can
function in mammalian cells (e.g. Kashani-Sabet et al., 1992 Antisense Res.
Dev., 2, 3-15; Ojwang
et al., 1992 Proc. Natl. Acad. Sci. U S A, 89, 10802-6; Chen et al., 1992
Nucleic Acids Res., 20,
4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci. U S A, 90, 6340-4; L'Huillier
et al., 1992 EMBO J.
11, 4411-8; Lisziewicz et al., 1993 Proc. Natl. Acad. Sci. U. S. A., 90, 8000-
4;Thompson et al.,
1995 Nucleic Acids Res. 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566).
The above
ribozyme transcription units can be incorporated into a variety of vectors for
introduction into
mammalian cells, including but not restricted to, plasmid DNA vectors, viral
DNA vectors (such as
adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as
retroviral or alphavirus
vectors) (for a review see Couture and Stinchcomb, 1996, supra).
By "highly conserved sequence region" is meant, a nucleotide sequence of one
or more
regions in a target gene does not vary significantly from one generation to
the other or from one
biological system to the other.
The nucleic acid-based inhibitors of the invention are added directly, or can
be complexed
with cationic lipids, packaged within liposomes, or otherwise delivered to
target cells or tissues. The
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
16
nucleic acid or nucleic acid complexes can be locally administered to relevant
tissues ex vivo, or in
vivo through injection, infusion pump or stmt, with or without their
incorporation in biopolymers.
In another aspect, the invention provides mammalian cells containing one or
more nucleic
acid molecules and/or expression vectors of this invention. The one or more
nucleic acid molecules
can independently be targeted to the same or different sites.
By "comprising" is meant including, but not limited to, whatever follows the
word
"comprising". Thus, use of the term "comprising" indicates that the listed
elements are required or
mandatory, but that other elements are optional and may or may not be present.
By "consisting of
is meant including, and limited to, whatever follows the phrase "consisting
of'. Thus, the phrase
"consisting of indicates that the listed elements are required or mandatory,
and that no other
elements may be present.
Other features and advantages of the invention will be apparent from the
following description
of the preferred embodiments thereof, and from the claims.
Description of the Preferred Embodiments
The drawings will first briefly be described.
Drawings:
Figure 1 is a diagram that shows the construct on which the DNA enzyme
selection is based.
Capital letters indicate the four standard DNA bases and RNA bases (reverse
type) in regions of the
molecular library which were constant among all molecules. These regions
provided the RNA
substrate, positioned the random region adjacent to the substrate by base-
pairing and allowed
manipulation of the library during selection using standard methods. Catalytic
sequences were
derived from the region of 50 nucleotides, imbedded between the constant
regions, synthesized to
contain random DNA sequences. Molecules were selected on the basis of their
ability to self cleave
at an RNA residue. Molecules were reacted in solution in a buffer which
approximated the
composition of Escherichia coli cytoplasm with respect to conditions expected
to be relevant to
RNA cleavage
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
17
Figure 2 is a diagram that shows a non-limiting secondary-structure model for
Clone 27 of a
class I DNA enzyme of the instant invention. The secondary structure shown in
Figure 2a is based
on the region of conserved nucleotides underlined in Table 2 with base pairing
restored at the 3' end
of the catalyst. The Ka for this enzyme interacting with substrate is 5 pM.
The enzyme does not
require glutathione but does lose 10-fold activity in the absence of
putrescine. Its pKa is ~ 7.9,
reaching a maximum pseudo first order rate constant in 0.5 mM Mg2+ of 0.06
miri'. The enzyme
also cleaves the in vitro transcribed target shown in Figure 2b, demonstrating
that it does not require
DNA residues in the substrate and can be generalized by changing the substrate
binding arms.
Figure 3 is a diagram showing a non-limiting secondary structure model for
clone 37 of a
class II DNA enzyme of the instant invention.
Figure 4 is a diagram that shows a selection scheme for the isolation of self
cleaving DNA
enzymes from a random-sequence DNA population.
Figure 5 is a diagram that shows the substrate sequence requirements of Class
I motif
nucleozymes of the invention.
Figure 6 is a diagram that shows the kinetic parameters of a Class I motif
nucleozyme of the
invention.
Figure 7 is a diagram showing proposed secondary structures of Class IV and
Class V trans-
cleaving nucleozymes of the invention.
Figure 8A is a diagram that shows a selection scheme for the isolation of self
cleaving
ribozymes from a random-sequence RNA population. (I) RNAs are incubated under
permissive
reaction conditions. (II) The 5' fragments of cleaved RNAs are separated from
uncleaved
precursors by denaturing 10% PAGE and recovered by crush/soaking from the
appropriate gel
section. (III) The recovered RNA fragments are amplified by RT-PCR, which
introduces a T7
promoter sequence and restores the nucleotides that were lost upon ribozyme
cleavage. The resulting
double-stranded DNAs are transcribed by T7 RNAP to generate the subsequent
population of RNAs.
Figure 8B is a diagram of the RNA construct used to initiate the in vitro
selection process. N4°
depicts 40 random-sequence positions. Underlined nucleotides identify the
region that represents all
16 possible nearest neighbor combinations.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
18
Figure 9 is a diagram that shows non-limiting secondary-structure models for
different classes
of self cleaving ribozymes of this invention. For each class (I-XII), the
generation of its first
appearance in the selection process (see Figure 8) is encircled. In addition,
the cleavage sites
(arrowheads) and observed rate constants are given for all RNA constructs as
depicted. In some
cases, secondary-structure models are based on artificial phylogenetic data
(e.g. see Table 11) or
otherwise reflect the most stable structure predicted by the Zuker RNA mfold
program that is
accessible on the Internet (www.ibc.wustl.edu/~mfold/rna/forml.c~i ). Putative
Watson/Crick base
pairing interactions are represented by dashes and putative G~U wobble
interactions are represented
by dots. Note that classes VI and VII were examined as unimolecular
constructs. No reasonable
secondary-structure model was established for class VII ribozymes. In this
case, the boxed regions
identify variable (shaded) or conserved nucleotides that reside in the
original random-sequence
domain. The nucleotides of the hammerhead ribozyme (class VIII) are numbered
according to the
system suggested by Hertel et al. (Hertel et al., 1992, Nucleic Acids
Research, 20, 3252).
Figure 10A is a diagram showing cleavage reaction profiles for bimolecular
class I (open
circles) and class II (filled circles) ribozymes as depicted in Figure 9 under
the permissive reaction
conditions. Observed rate constants for class I and class II ribozymes are
0.01 and 0.05 miri',
respectively, as determined by the negative slope of the lines. Figure lOB
shows a compilation of
the cleavage sites of the 12 classes of ribozymes. Numbers identify the
nucleotides within the
nearest-neighbor domain depicted in Figure 8B.
Figure 11 shows a comparison of the secondary structures of two hammerhead
ribozymes and
the dominant X motif. Figure 11A is a diagram of two distinct versions (i and
ii) of the hammerhead
ribozyme which were isolated and examined for catalytic activity with S21
substrate. Both variants
retain the highly conserved catalytic core, which is known to tolerate
sequence variation only at
position 7 (Tang and Breaker, 1997, RNA, 3, 914-925). Stem elements and
nucleotides in (~ are
numbered according to the nomenclature defined by Hertel et al. (Hertel et
al., supra). Figure 11B
shows the dominant unimolecular construct isolated after a total of 25 rounds
of in vitro selection
conforms to the X-motif (class I) class of self cleaving ribozymes. This self
cleaving ribozyme can
be reorganized into a bimolecular format wherein separate substrate RNAs (S21)
are cleaved by a
43-nt enzyme domain, the latter which encompasses all highly conserved
nucleotides that were
identified during reselection (Table 11). Enzyme-substrate interactions result
from the formation of
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
19
two (stems I and IV) of the four putative helical regions that define the
motif. Arrowheads identify
the sites of ribozyme-mediated cleavage.
Figure 12 is a demonstration of cleavage site versatility of the X motif
ribozyme. Figure 12
shows three different 43-nt RNAs carrying the conserved core of the X motif
class of ribozymes
were generated by in vitro transcription such that each differed in the base
pairing potential of
binding arms I and IV. Specifically, ribozyme X-E1 is engineered to form eight
base pairs that flank
both the 5' and 3' sides of an unpaired G reside. The lines represent
nucleotides within binding arms
I and IV that are complementary to the X-E1 target site (depicted by the
arrow). Similarly,
ribozymes X-E2 and X-E3 carry the corresponding binding arm sequences that
allow base pairing
only with their corresponding target sites. Base pairing interactions are
depicted by dashes.
Figure 13 is a representative diagram of a Class V ribozyme motif which shows
the effect of
sequential 2'-O-methyl substitutions on Kobs in the ribozyme core. Figure 13
also shows a typical
site of substrate cleavage for the Class V ribozyme as well as the numbering
system used in this
application for describing modifications to this class of ribozyme as
described in Tables VI-IX.
Figure 14 is a representative diagram of structural similarities between Class
I (SEQ ID NO
146), (substrate SEQ ID NO 147) and Class VIII (SEQ ID NO 148), (substrate SEQ
m NO 149)
enzymatic nucleic acid molecules of the invention.
Figure 15 is a comparison of the kinetic characteristics of the Class I and
Class VIII
enzymatic nucleic acid molecules of the invention. (A) Saturation of substrate
with enzymes based
on Class I motif and Class VIII motif enzymes, respectively. (B) Influence of
monovalent ions on
ribozyme activity. (C) Magnesium dependence of each ribozyme. Reactions were
conducted in 50
mM Tris-HC1 (pH 7.5 at 23°C), and 20 mM magnesium chloride unless
otherwise indicated.
Nucleic Acid Catalysts
The invention provides nucleic acid catalysts and methods for producing a
class of enzymatic
nucleic acid cleaving agents which exhibit a high degree of specificity for
the nucleic acid sequence
of a desired target. The enzymatic nucleic acid molecule is preferably
targeted to a highly conserved
sequence region of a target such that specific diagnosis and/or treatment of a
disease or condition in
a variety of biological systems can be provided with a single enzymatic
nucleic acid. Such
enzymatic nucleic acid molecules can be delivered exogenously to specific
cells as required. In the
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
preferred Class I and II motifs, the small size (less than 60 nucleotides,
preferably between 25-40
nucleotides in length) of the molecule allows the cost of treatment to be
reduced.
By "nucleic acid catalyst" as used herein is meant a nucleic acid molecule
(e.g., the molecules
of formulae I, II, III and IV), capable of catalyzing (altering the velocity
and/or rate of) a variety of
5 reactions including the ability to repeatedly cleave other separate nucleic
acid molecules
(endonuclease activity) in a nucleotide base sequence-specific manner. Such a
molecule with
endonuclease activity may have complementarity in a substrate binding region
(e.g. X and Y in
formulae I, II, III, and IV) to a specified gene target, and also has an
enzymatic activity that
specifically cleaves RNA or DNA in that target. That is, the nucleic acid
molecule with
10 endonuclease activity is able to intramolecularly or intermolecularly
cleave RNA or DNA and
thereby inactivate a target RNA or DNA molecule. This complementarity
functions to allow
sufficient hybridization of the enzymatic RNA molecule to the target RNA or
DNA to allow the
cleavage to occur. 100% complementarity is preferred, but complementarity as
low as 50-75% may
also be useful in this invention. The nucleic acids may be modified at the
base, sugar, and/or
15 phosphate groups. The term enzymatic nucleic acid is used interchangeably
with phrases such as
ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, nucleozyme, RNA
enzyme,
endoribonuclease, endonuclease, minizyme, oligozyme, finderon or nucleic acid
catalyst. All of
these terminologies describe nucleic acid molecules with enzymatic activity.
The specific examples
of enzymatic nucleic acid molecules described in the instant application are
not limiting in the
20 invention and those skilled in the art will recognize that all that is
important in an enzymatic nucleic
acid molecule of this invention is that it has a specific substrate binding
site which is complementary
to one or more of the target nucleic acid regions, and that it have nucleotide
sequences within or
surrounding that substrate binding site which impart a nucleic acid cleaving
activity to the molecule.
In preferred embodiments of the present invention, an enzymatic nucleic acid
molecule, is 13
to 100 nucleotides in length, e.g., in specific embodiments 35, 36, 37, or 38
nucleotides in length
(e.g., for particular DNA enzymes). In particular embodiments, the nucleic
acid molecule is 15-100,
17-100, 20-100, 21-100, 23-100, 25-100, 27-100, 30-100, 32-100, 35-100, 40-
100, 50-100, 60-100,
70-100, or 80-100 nucleotides in length. Instead of 100 nucleotides being the
upper limit on the
length ranges specified above, the upper limit of the length range can be, for
example, 30, 40, 50, 60,
70, or 80 nucleotides. Thus, for any of the length ranges, the length range
for particular
embodiments has lower limit as specified, with an upper limit as specified
which is greater than the
lower limit. For example, in a particular embodiment, the length range can be
35-50 nucleotides in
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
21
length. All such ranges are expressly included. Also in particular
embodiments, a nucleic acid
molecule can have a length which is any of the lengths specified above, for
example, a catalytic core
of 21 conserved nucleotides in length with variable length binding arms and/or
variable regions.
The enzymatic nucleic acid molecules of Formulae 1, II, III and IV can
independently
comprise a cap structure which can independently be present or absent.
By "chimeric nucleic acid molecule" or "mixed polymer" is meant that, the
molecule can be
comprised of both modified or unmodified nucleotides.
By "cap structure" is meant chemical modifications, which have been
incorporated at either
terminus of the oligonucleotide. These terminal modifications protect the
nucleic acid molecule from
exonuclease degradation, and can help in delivery and/or localization within a
cell. The cap can be
present at the 5'-terminus (S'-cap) or at the 3'-terminus (3'-cap) or can be
present on both termini.
In non-limiting examples the S'-cap is selected from the group consisting of
the following: inverted
abasic residue (moiety); 4',5'-methylene nucleotide; 1-(beta-D-
erythrofuranosyl) nucleotide, 4'-thio
nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-
nucleotides; alpha-nucleotides;
modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl
nucleotide; acyclic
3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-
dihydroxypentyl nucleotide,
3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted
nucleotide moiety; 3'-2'-
inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate;
hexylphosphate; aminohexyl
phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; or bridging
or non-bridging
methylphosphonate moiety (for more details, see Beigelman et al.,
International PCT publication
No. WO 97/26270, incorporated by reference herein).
In yet another preferred embodiment, the 3'-cap is selected from a group
consisting of the
following: 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide;
4'-thio nucleotide;
carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl
phosphate, 3-aminopropyl
phosphate; G-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-
anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base
nucleotide;
phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco.
nucleotide; 3,4-
dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide; 5'-5'-inverted
nucleotide moiety; 5'-S'-
inverted abasic moiety; S'-phosphoramidate; 5'-phosphorothioate; 1,4-
butanediol phosphate; S'-
amino; bridging and/or non-bridging S'-phosphoramidate; phosphorothioate
and/or
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
22
phosphorodithioate; bridging or non bridging methylphosphonate; and 5'-
mercapto moieties (for
more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated
by reference herein).
By "oligonucleotide" as used herein, is meant a molecule comprising two or
more nucleotides.
The specific enzymatic nucleic acid molecules described in the instant
application are not
limiting in the invention and those skilled in the art will recognize that all
that is important in an
enzymatic nucleic acid molecule of this invention is that it has a specific
substrate binding site (e.g.,
X and Y of Formulae I, II, III and IV above) which is complementary to one or
more of the target
nucleic acid regions, and that it have nucleotide sequences within or
surrounding that substrate
binding site which impart a nucleic acid cleaving activity to the molecule.
The determination of whether the V or L regions can be deleted or changed can
be determined
by experimentation and tested in vitro using the methods described herein.
Similarly, when the V
region is present the determination or whether its length can be increased or
decreased can be
evaluated using the selection protocols described herein.
By "enzymatic portion" is meant that part of the enzymatic nucleic acid
molecule essential for
cleavage of an RNA substrate.
By "substrate binding arm" or "substrate binding domain" is meant that portion
of a enzymatic
nucleic acid molecule which is complementary to (i.e., able to base-pair with)
a portion of its
substrate. Generally, such complementarity is 100%, but can be less if
desired. For example, as few
as 10 nucleotides out of a total of 14 can be base-paired. Such arms are shown
generally in Figures
2, 3, 7, 9, and 11 and as X and Y in Formulae I - IV. That is, these arms
contain sequences within
an enzymatic nucleic acid molecule which are intended to bring enzymatic
nucleic acid molecule
and target RNA together through complementary base-pairing interactions. The
enzymatic nucleic
acid molecules such as ribozymes and DNAzymes of the invention can have
binding arms that are
contiguous or non-contiguous and can be of varying lengths. The length of the
binding arms) are
preferably greater than or equal to four nucleotides; specifically 12-100
nucleotides; more
specifically 14-24 nucleotides long. The two binding arms are chosen, such
that the length of the
binding arms are symmetrical (i.e., each of the binding arms is of the same
length; e.g., five and five
nucleotides, six and six nucleotides or seven and seven nucleotides long) or
asymmetrical (i.e., the
binding arms are of different length; e.g., six and three nucleotides; three
and six nucleotides long;
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
23
four and five nucleotides long; four and six nucleotides long; four and seven
nucleotides long; and
the like).
Catalytic activity of the enzymatic nucleic acid molecule described in the
instant invention
can be optimized as described by Usman et al., US Patent No. 5,807,718. The
methods described by
Draper et al., supra, for nucleic acid catalysts can readily be applied for
use in the optimization of
the nucleic acid molecules of the instant invention. Specific details will not
be repeated here, but
include altering the length of the enzymatic nucleic acid molecule binding
arms, or chemically
synthesizing enzymatic nucleic acid molecules with modifications (base, sugar
and/or phosphate)
that prevent their degradation by serum ribonucleases and/or enhance their
enzymatic activity (see
e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et
al., 1990 Nature 344,
565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in
Biochem. Sci. 17,
334; Usman et al., International Publication No. WO 93/15187; Rossi et al.,
International
Publication No. WO 91/03162; Sproat, US Patent No. 5,334,711; and Burgin et
al., supra; all of
these describe various chemical modifications that can be made to the base,
phosphate and/or sugar
moieties of enzymatic DNA and/or RNA molecules). All these publications are
hereby incorporated
by reference herein. Modifications which enhance their efficacy in cells, and
removal of bases from
stem loop structures to shorten enzymatic nucleic acid molecule synthesis
times and reduce chemical
requirements are desired. The enzymatic nucleic acid molecules can be
synthesized entirely of
deoxyribonucleotides, or other 2'-modified nucleotides (e.g.; 2'-O-methyl, 2'-
O-allyl, 2'-C-allyl, 2'-
deoxy-2'-amino, 2'-deoxy-2'-fluoro, 2'-O-amino etc.), individually or in
combination, so long as the
nucleic acid catalyst is functional.
Therapeutic enzymatic nucleic acid molecules should remain stable within cells
until
translation of the target RNA has been inhibited long enough to reduce the
levels of the undesirable
protein. This period of time varies between hours to days depending upon the
disease state. In
particular, DNAzymes should be resistant to nucleases in order to function as
effective intracellular
therapeutic agents. Improvements in the chemical synthesis of DNA and RNA
(Wincott et al., 1995
Nucleic Acids Res. 23, 2677; incorporated by reference herein) have expanded
the ability to modify
DNA enzymes to enhance their nuclease stability.
By "nucleotide" is meant a heterocyclic nitrogenous base in N-glycosidic
linkage with a
phosphorylated sugar. Nucleotides are recognized in the art to include natural
bases (standard), and
modified bases well known in the art. Such bases are generally located at the
1' position of a
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
24
nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a
phosphate group. The
nucleotides can be unmodified or modif ed at the sugar, phosphate and/or base
moiety, (also referred
to interchangeably as nucleotide analogs, modified nucleotides, non-natural
nucleotides, non-
standard nucleotides and other; see for example, Usman and McSwiggen, supra;
Eckstein et al.,
International PCT Publication No. WO 92/07065; Usman et al., International PCT
Publication No.
WO 93/15187; Uhlman & Peyman, supra all are hereby incorporated by reference
herein). There are
several examples of modified nucleic acid bases known in the art as summarized
by Limbach et al.,
1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of
chemically modified and
other natural nucleic acid bases that can be introduced into nucleic acids
include, inosine, purine,
pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy
benzene, 3-methyl uracil,
dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-
methylcytidine), 5-alkyluridines
(e.g., ribothymidine), S-halouridine (e.g., 5-bromouridine) or 6-
azapyrimidines or 6-
alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-
thiouridine,
wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine,
S'-
carboxymethylaminomethyl-2-thiouridine, S-carboxymethylaminomethyluridine,
beta-D-
galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,
3-methylcytidine,
2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-
methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-
methylcarbonylmethyluridine,
5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-
isopentenyladenosine, ?-D-
mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine
derivatives and others
(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By
"modified bases" in
this aspect is meant nucleotide bases other than adenine, guanine, cytosine
and uracil at 1' position
or their equivalents; such bases can be used at any position, for example,
within the catalytic core of
an enzymatic nucleic acid molecule and/or in the substrate-binding regions of
the nucleic acid
molecule.
In a preferred embodiment, the invention features modified DNAzymes with
phosphate
backbone modifications comprising one or more phosphorothioate,
phosphorodithioate,
methylphosphonate, morpholino, amidate carbamate, carboxymethyl, acetamidate,
polyamide,
sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or
alkylsilyl, substitutions. For a
review of oligonucleotide backbone modifications see Hunziker and Leumann,
1995, Nucleic Acid
Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-
417, and
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in
Carbohydrate
Modifications in Antisense Research, ACS, 24-39.
In a further preferred embodiment of the instant invention, an inverted deoxy
abasic moiety is
utilized at the 3' end of the enzymatic nucleic acid molecule.
5 In particular, the invention features modified enzymatic nucleic acid
molecules having a base
substitution selected from pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,
2, 4, 6-trimethoxy
benzene, 3-methyluracil, dihydrouracil, naphthyl, 6-methyl-uracil and
aminophenyl.
There are several examples in the art describing sugar and phosphate
modifications that can be
introduced into enzymatic nucleic acid molecules without significantly
effecting catalysis and with
10 significant enhancement in their nuclease stability and efficacy. Enzymatic
nucleic acid molecules
are modified to enhance stability and/or enhance catalytic activity by
modification with nuclease
resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl,
nucleotide base
modifications (for a review see Usman and Cedergren, 1992 TIES 17, 34; Usman
et al., 1994
Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996 Biochemistry 35, 14090).
Sugar modifications
15 of enzymatic nucleic acid molecules have been extensively described in the
art (see Eckstein et al.,
International Publication PCT No. WO 92/07065; Perrault et al. Nature 1990,
344, 565-568;
Pieken et al. Science 1991, 253, 314-317; Usman and Cedergren, Trends in
Biochem. Sci. 1992, 17,
334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat,
US Patent No.
5,334,711 and Beigelman et al., 1995 J. Biol. Chem. 270, 25702).
20 Additionally, the enzymatic nucleic acid molecules can be linked to various
chemical moieties
and/or ligands to enhance stability, localization, and/or efficacy. Such
moieties and ligands include
but are not limited to polyethylene glycol (PEG), cholesterol, cytofectins
(such as DOPE, DDAB,
DOGS, DOTMA and DOTMA analogues including DOTAP, DMRIE, DOSPA, DORIE, DORI,
and
GAP-DLRIE), glucose, galactose, spermine, spermidine, C-dextran,
polyacrylamide, biotin, retinoic
25 acid, peptide nucleic acids, antigens (such as CD40, CD44,
carcinoembryonic, endoglin, and
prostate-specific antigens), receptors (such as VEGF, HER2/neu), and other
fatty acids, steroids,
cationic lipids, polyamines, polyamides, glucocorticoids, integrins, histones,
protamines, toxins,
viroids, virusoids, amino acids, peptides, proteins, sugars, polysaccharides,
glycoconjugates,
oligonucleotides, metals, small molecules, macromolecules and combinations
thereof. For a review
of non-limiting carbohydrate modifications including 2'-conjugates, see
Manoharan, 1999, Biochim.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
26
Biophys. Acta., 1489(1), 117-130. For a review of non-limiting drug
macromolecule conjugates, see
Takakura et al., 1996, Advan. Drug, Delivery Rev., 19(3), 377-399. Such
publications describe
general methods and strategies to determine the location of incorporation of
sugar, base and/or
phosphate modifications and the like into nucleic acid molecules without
inhibiting catalysis, and
are incorporated by reference herein. In view of such teachings, similar
modifications can be used
as described herein to modify the nucleic acid catalysts of the instant
invention.
As the term is used in this application, non-nucleotide-containing enzymatic
nucleic acid
means a nucleic acid molecule that contains at least one non-nucleotide
component which replaces a
portion of an enzymatic nucleic acid molecule, e.g., but not limited to, a
double-stranded stem, a
single-stranded "catalytic core" sequence, a single-stranded loop or a single-
stranded recognition
sequence. These molecules are able to cleave (preferably, repeatedly cleave
through multiple
turnover) separate RNA or DNA molecules in a nucleotide base sequence specific
manner. Such
molecules can also act to cleave intramolecularly if that is desired. Such
enzymatic molecules can
be targeted to virtually any RNA transcript.
The sequences of enzymatic nucleic acid molecules that are chemically
synthesized, useful in
this invention, are shown in the Tables and Figures. Those in the art will
recognize that these
sequences are representative only of many more such sequences where the
enzymatic portion of the
enzymatic nucleic acid molecule (all but the binding arms) is altered to
affect activity. The
enzymatic nucleic acid molecule sequences listed in the tables and figures can
be formed of
deoxyribonucleotides or other nucleotides or non-nucleotides. Such enzymatic
nucleic acid
molecules with enzymatic activity are equivalent to the enzymatic nucleic acid
molecules described
specifically in the tables and figures.
Synthesis of Nucleic Acid Molecules
Synthesis of nucleic acids greater than 100 nucleotides in length can be
difficult using
automated methods, and the therapeutic cost of such molecules can be
prohibitive. In this invention,
small nucleic acid motifs ("small" refers to nucleic acid motifs no more than
100 nucleotides in
length, preferably no more than 80 nucleotides in length, and most preferably
no more than SO
nucleotides in length; e.g., antisense oligonucleotides, hammerhead or the
Inozyme enzymatic
nucleic acids) are preferably used for exogenous delivery. The simple
structure of these molecules
increases the ability of the nucleic acid to invade targeted regions of RNA
structure. Exemplary
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
27
molecules of the instant invention are chemically synthesized, and others can
similarly be
synthesized.
The nucleic acid molecules of the invention, including certain enzymatic
nucleic acid
molecules, can be synthesized using the methods described in Usman et al.,
1987, J. Am. Chem.
Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and
Wincott et al., 1995,
Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74,
59. Such methods
make use of common nucleic acid protecting and coupling groups, such as
dimethoxytrityl at the 5'-
end, and phosphoramidites at the 3'-end. In a non-limiting example, small
scale syntheses are
conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 ~mol scale
protocol with a 7.5
min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling
step for 2'-O-
methylated nucleotides. Table 1 outlines the amounts and the contact times of
the reagents used in
the synthesis cycle. Alternatively, syntheses at the 0.2 ~mol scale can be
done on a 96-well plate
synthesizer, such as the PG2100 instrument produced by Protogene (Palo Alto,
CA) with minimal
modification to the cycle. A 33-fold excess (60 ~,L of 0.11 M = 6.6 ~,mol) of
2'-O-methyl
phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 pL of 0.25 M =
15 ~mol) can be used
in each coupling cycle of 2'-O-methyl residues relative to polymer-bound S'-
hydroxyl. A 66-fold
excess (120 ~L of 0.11 M = 13.2 pmol) of alkylsilyl (ribo) protected
phosphoramidite and a 150-
fold excess of S-ethyl tetrazole (120 pL of 0.25 M = 30 pmol) can be used in
each coupling cycle of
ribo residues relative to polymer-bound 5'-hydroxyl. Average coupling yields
on the 394 Applied
Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the
trityl fractions, are
typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394
Applied Biosystems, Inc.
synthesizer include; detritylation solution is 3% TCA in methylene chloride
(ABI); capping is
performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic
anhydride/10% 2,6-lutidine
in THF (ABI); oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in
THF
(PERSEPTIVETM). Burdick & Jackson Synthesis Grade acetonitrile is used
directly from the
reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up
from the solid obtained
from American International Chemical, Inc. Alternately, for the introduction
of phosphorothioate
linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in
acetonitrile) is used.
Cleavage from the solid support and deprotection of the oligonucleotide is
typically performed
using either a two-pot or one-pot protocol. For the two-pot protocol, the
polymer-bound trityl-on
oligoribonucleotide is transferred to a 4 mL glass screw top vial and
suspended in a solution of 40%
aq. methylamine (1 mL) at 65 °C for 10 min. After cooling to -20
°C, the supernatant is removed
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
28
from the polymer support. The support is washed three times with 1.0 mL of
EtOH:MeCN:H20/3:1:1, vortexed and the supernatant is then added to the first
supernatant. The
combined supernatants, containing the oligoribonucleotide, are dried to a
white powder. The base
deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP
solution (300 pL of a
solution of 1.5 mL N-methylpyrrolidinone, 750 ~L TEA and 1 mL TEA~3HF to
provide a 1.4 M HF
concentration) and heated to 65 °C. After 1.5 h, the oligomer is
quenched with 1.5 M NH4HC03.
Alternatively, for the one-pot protocol, the polymer-bound trityl-on
oligoribonucleotide is
transferred to a 4 mL glass screw top vial and suspended in a solution of 33%
ethanolic
methylamine/DMSO: 1/1 (0.8 mL) at 65°C for 15 min. The vial is brought
to r.t. TEA~3HF (0.1
mL) is added and the vial is heated at 65 °C for 1 S min. The sample is
cooled at -20 °C and then
quenched with 1.5 M NH4HC03. An alternative deprotection cocktail for use in
the one pot
protocol comprises the use of aqueous methylamine (0.5 ml) at 65°C for
15 min followed by DMSO
(0.8 ml) and TEA~3HF (0.3 ml) at 65°C for 15 min. A similar methodology
can be employed with
96-well plate synthesis formats by using a Robbins Scientific Flex Chem block,
in which the
reagents are added for cleavage and deprotection of the oligonucleotide.
For anion exchange desalting of the deprotected oligomer, the TEAB solution is
loaded onto a
Qiagen 500~ anion exchange cartridge (Qiagen Inc.) that is prewashed with 50
mM TEAB (10 mL).
After washing the loaded cartridge with 50 mM TEAB (10 mL), the RNA is eluted
with 2 M TEAB
( 10 mL) and dried down to a white powder.
For purification of the trityl-on oligomers, the quenched NH4HC03 solution is
loaded onto a
C-18 containing cartridge that had been prewashed with acetonitrile followed
by 50 mM TEAA.
After washing the loaded cartridge with water, the RNA is detritylated with
0.5% TFA for 13 min.
The cartridge is then washed again with water, salt exchanged with 1 M NaCI
and washed with
water again. The oligonucleotide is then eluted with 30% acetonitrile.
Alternatively, for
oligonucleotides synthesized in a 96-well format, the crude trityl-on
oligonucleotide is purified
using a 96-well solid phase extraction block packed with C 18 material, on a
Bohdan Automation
workstation.
The average stepwise coupling yields are typically >98% (Wincott et al., 1995
Nucleic Acids
Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that
the scale of synthesis can
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
29
be adapted as larger or smaller than the example described above including but
not limited to 96
well format, all that is important is the ratio of chemicals used in the
reaction.
To ensure the quality of synthesis of nucleic acid molecules of the invention,
quality control
measures are utilized for the analysis of nucleic acid material. Capillary Gel
Electrophoresis, for
example using a Beckman MDQ CGE instrument, can be ulitized for rapid analysis
of nucleic acid
molecules, by introducing sample on the short end of the capillary. In
addition, mass spectrometry,
for example using a PE Biosystems Voyager-DE MALDI instrument, in combination
with the
Bohdan workstation, can be utilized in the analysis of oligonucleotides,
including oligonucleotides
synthesized in the 96-well format.
Enzymatic nucleic acids can also be synthesized in two parts and annealed to
reconstruct the
active enzymatic nucleic acid (Chowrira and Burke, 1992 Nucleic Acids Res.,
20, 2835-2840).
Enzymatic nucleic acids are also synthesized from DNA templates using
bacteriophage T7 RNA
polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51).
Alternatively, the nucleic acid molecules of the present invention can be
synthesized
separately and joined together post-synthetically, for example by ligation
(Moore et al., 1992,
Science 256, 9923; Draper et al., International PCT publication No. WO
93/23569; Shabarova et al.,
1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides &
Nucleotides, 16, 951;
Bellon et al., 1997, Bioconjugate Chem. 8, 204).
The nucleic acid molecules of the present invention are preferably modified to
enhance
stability by modification with nuclease resistant groups, for example, 2'-
amino, 2'-C-allyl, 2'-flouro,
2'-O-methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34;
Usman et al., 1994,
Nucleic Acids Symp. Ser. 31, 163). Enzymatic nucleic acids are purified by gel
electrophoresis
using known methods or are purified by high pressure liquid chromatography
(HPLC; See Wincott
et al., Supra, the totality of which is hereby incorporated herein by
reference) and are re-suspended
in water.
Administration of Nucleic Acid Molecules
Sullivan et al., PCT WO 94/02595, describe the general methods for delivery of
enzymatic
nucleic acid molecules. DNAzymes can be administered to cells by a variety of
methods known to
those familiar to the art, including, but not restricted to, encapsulation in
liposomes, by
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
iontophoresis, or by incorporation into other vehicles, such as hydrogels,
cyclodextrins,
biodegradable nanocapsules, and bioadhesive microspheres. For some
indications, DNAzymes can
be directly delivered ex vivo to cells or tissues with or without the
aforementioned vehicles.
Alternatively, the DNAzymes/vehicle combination is locally delivered by direct
injection or by use
5 of a catheter, infusion pump or stmt. Other routes of delivery include, but
are not limited to,
intravascular, intramuscular, subcutaneous or joint injection, aerosol
inhalation, oral (tablet or pill
form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.
More detailed
descriptions of nucleic acid molecule delivery and administration are provided
in Sullivan et al.,
supra and Draper et al., PCT W093/23569 which have been incorporated by
reference herein.
10 The molecules of the instant invention can be used as pharmaceutical
agents. Pharmaceutical
agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some
extent, preferably all of
the symptoms) of a disease state in a patient.
The negatively charged polynucleotides of the invention can be administered
(e.g., RNA,
DNA or protein) and introduced into a patient by any standard means, with or
without stabilizers,
15 buffers, and the like, to form a pharmaceutical composition. When it is
desired to use a liposome
delivery mechanism, standard protocols for formation of liposomes can be
followed. The
compositions of the present invention may also be formulated and used as
tablets, capsules or elixirs
for oral administration, suppositories for rectal administration, sterile
solutions, suspensions for
injectable administration, and other compositions known in the art.
20 The present invention also includes pharmaceutically acceptable
formulations of the
compounds described. These formulations include salts of the above compounds,
e.g., acid addition
salts, including salts of hydrochloric, hydrobromic, acetic acid, and benzene
sulfonic acid.
A pharmacological composition or formulation refers to a composition or
formulation in a form
suitable for administration, e.g., systemic administration, into a cell or
patient, preferably a human.
25 Suitable forms, in part, depend upon the use or the route of entry, for
example, oral, transdermal, or
by injection. Such forms should not prevent the composition or formulation
from reaching a target
cell (i.e., a cell to which the negatively charged polymer is desired to be
delivered to). For example,
pharmacological compositions injected into the blood stream should be soluble.
Other factors are
known in the art, and include considerations such as toxicity and forms which
prevent the
30 composition or formulation from exerting its effect.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
31
By "systemic administration" is meant in vivo systemic absorption or
accumulation of drugs in
the blood stream followed by distribution throughout the entire body.
Administration routes which
lead to systemic absorption include, without limitations: intravenous,
subcutaneous, intraperitoneal,
inhalation, oral, intrapulmonary and intramuscular. Each of these
administration routes expose the
desired negatively charged polymers, e.g., nucleic acids, to an accessible
diseased tissue. The rate
of entry of a drug into the circulation has been shown to be a function of
molecular weight or size.
The use of a liposome or other drug carrier comprising the compounds of the
instant invention can
potentially localize the drug, for example, in certain tissue types, such as
the tissues of the reticular
endothelial system (RES). A liposome formulation which can facilitate the
association of drug with
the surface of cells, such as, lymphocytes and macrophages is also useful.
This approach can
provide enhanced delivery of the drug to target cells by taking advantage of
the specificity of
macrophage and lymphocyte immune recognition of abnormal cells, such as cancer
cells.
The invention also features the use of a composition comprising surface-
modified liposomes
containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating
liposomes or stealth
liposomes). These formulations offer a method for increasing the accumulation
of drugs in target
tissues. This class of drug carriers resists opsonization and elimination by
the mononuclear
phagocytic system (MPS or RES), thereby enabling longer blood circulation
times and enhanced
tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95,
2601-2627; Ishiwata et
al., Chem. Pharm. Bull. 1995, 43, 1005-1011). The long-circulating liposomes
enhance the
pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to
conventional
cationic liposomes which are known to accumulate in tissues of the MPS (Liu et
al., J. Biol. Chem.
1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO
96/10391; Ansell et al.,
International PCT Publication No. WO 96/10390; Holland et al., International
PCT Publication No.
WO 96/10392; all of which are incorporated by reference herein). Long-
circulating liposomes are
also likely to protect drugs from nuclease degradation to a greater extent
compared to cationic
liposomes, based on their ability to avoid accumulation in metabolically
aggressive MPS tissues
such as the liver and spleen. Such liposomes have been shown to accumulate
selectively in tumors,
presumably by extravasation and capture in the neovascularized target tissues
(Lasic et al., Science
1995, 267, 1275-1276; Oku et a1.,1995, Biochim. Biophys. Acta, 1238, 86-90).
The present invention also includes compositions prepared for storage or
administration which
include a pharmaceutically effective amount of the desired compounds in a
pharmaceutically
acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic
use are well known in
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
32
the pharmaceutical art, and are described, for example, in Remington's
Pharmaceutical Sciences,
Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incorporated by reference
herein. For
example, preservatives, stabilizers, dyes and flavoring agents can be added to
the compositions. Id.
at 1449. Suitable examples include sodium benzoate, sorbic acid and esters of
p-hydroxybenzoic
acid. In addition, antioxidants and suspending agents can be added to the
compositions.
A pharmaceutically effective dose is that dose required to prevent, inhibit
the occurrence, or
treat (alleviate a symptom to some extent, preferably all of the symptoms) of
a disease state. The
pharmaceutically effective dose depends on the type of disease, the
composition used, the route of
administration, the type of mammal being treated, the physical characteristics
of the specific
mammal under consideration, concurrent medication, and other factors which
those skilled in the
medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100
mg/kg body
weight/day of active ingredients is administered dependent upon potency of the
negatively charged
polymer. In a one aspect, the invention provides enzymatic nucleic acid
molecules that can be
delivered exogenously to specific cells as required. The enzymatic nucleic
acid molecules are added
directly, or can be complexed with cationic lipids, packaged within liposomes,
or otherwise
delivered to smooth muscle cells. The RNA or RNA complexes can be locally
administered to
relevant tissues through the use of a catheter, infusion pump or stmt, with or
without their
incorporation in biopolymers. Using the methods described herein, other
enzymatic nucleic acid
molecules that cleave target nucleic acid may be derived and used as described
above. Specific
examples of nucleic acid catalysts of the instant invention are provided below
in the Tables and
Figures.
Alternatively, the enzymatic nucleic acid molecules of the instant invention
can be expressed
within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985
Science 229, 345; McGarry
and Lindquist, 1986 Proc. Natl. Acad. Sci. USA 83, 399; Scanlon et al., 1991,
Proc. Natl. Acad. Sci.
USA, 88, 10591-S; Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15;
Dropulic et al., 1992 J.
Virol, 66, 1432-41; Weerasinghe et al., 1991 J. Virol, 65, 5531-4; Ojwang et
al., 1992 Proc. Natl.
Acad. Sci. USA 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9;
Sarver et al.,
1990 Science 247, 1222-1225; Thompson et al., 1995 Nucleic Acids Res. 23,
2259; Good et al.,
1997, Gene Therapy, 4, 45; all of the references are hereby incorporated in
their totality by reference
herein). Those skilled in the art realize that any nucleic acid can be
expressed in eukaryotic cells
from the appropriate DNA/RNA vector. The activity of such nucleic acids can be
augmented by
their release from the primary transcript by a enzymatic nucleic acid (Draper
et al., PCT WO
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
33
93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992 Nucleic
Acids Symp. Ser.,
27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al.,
1993 Nucleic Acids
Res., 21, 3249-55; Chowrira et al., 1994 J. Biol. Chem. 269, 25856; all of the
references are hereby
incorporated in their totality by reference herein).
In another aspect of the invention, enzymatic nucleic acid molecules that
cleave target
molecules are expressed from transcription units (see for example Couture et
al., 1996, TIG., 12,
S 10) inserted into DNA or RNA vectors. The recombinant vectors are preferably
DNA plasmids or
viral vectors. Enzymatic nucleic acid expressing viral vectors could be
constructed based on, but not
limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.
Preferably, the recombinant
vectors capable of expressing the enzymatic nucleic acids are delivered as
described above, and
persist in target cells. Alternatively, viral vectors can be used that provide
for transient expression of
enzymatic nucleic acids. Such vectors can be repeatedly administered as
necessary. Once expressed,
the enzymatic nucleic acids cleave the target mRNA. The active enzymatic
nucleic acid contains an
enzymatic center or core equivalent to those in the examples, and binding arms
able to bind target
nucleic acid molecules such that cleavage at the target site occurs. Other
sequences can be present
which do not interfere with such cleavage. Delivery of enzymatic nucleic acid
expressing vectors
can be systemic, such as by intravenous or intramuscular administration, by
administration to target
cells ex-planted from the patient followed by reintroduction into the patient,
or by any other means
that allows for introduction into the desired target cell (for a review see
Couture et al., 1996, TIG.,
12, 510).
In one aspect the invention features, an expression vector comprising nucleic
acid sequence
encoding at least one of the nucleic acid catalyst of the instant invention is
disclosed. The nucleic
acid sequence encoding the nucleic acid catalyst of the instant invention is
operably linked in a
manner which allows expression of that nucleic acid molecule.
In another aspect the invention features an expression vector comprising: a) a
transcription
initiation region (e.g., eukaryotic pol 1, II or III initiation region); b) a
transcription termination
region (e.g., eukaryotic pol I, II or III termination region); c) a nucleic
acid sequence encoding at
least one of the nucleic acid catalyst of the instant invention; and wherein
said sequence is operably
linked to said initiation region and said termination region, in a manner
which allows expression
and/or delivery of said nucleic acid molecule. The vector may optionally
include an open reading
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
34
frame (ORF) for a protein operably linked on the 5' side or the 3'-side of the
sequence encoding the
nucleic acid catalyst of the invention; and/or an intron (intervening
sequences).
Transcription of the nucleic acid molecule sequences can be driven from a
promoter for
eukaryotic RNA polymerise I (pol I), RNA polymerise II (pol II), or RNA
polymerise III (pol III).
Transcripts from pol II or pol III promoters are generally expressed at high
levels in all cells; the
levels of a given pol II promoter in a given cell type will depend on the
nature of the gene regulatory
sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA
polymerise promoters can
be also used, providing that the prokaryotic RNA polymerise enzyme is
expressed in the appropriate
cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acid. Sci. U S A, 87, 6743-7;
Gao and Huang 1993,
Nucleic Acids Res.., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217,
47-66; Zhou et al.,
1990, Mol. Cell. Biol., 10, 4529-37). All of these references are incorporated
by reference herein.
Several investigators have demonstrated that nucleic acid molecules, such as
enzymatic nucleic
acids, expressed from such promoters function in mammalian cells (e.g. Kashani-
Sabet et al., 1992,
Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acid. Sci. U S
A, 89, 10802-6;
Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc.
Natl. Acid. Sci. U S A,
90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al.,
1993, Proc. Natl.
Acid. Sci. U. S. A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23,
2259; Sullenger &
Cech, 1993, Science, 262, 1566). More specifically, transcription units such
as the ones derived
from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and
adenovirus VA RNA
are useful in generating high concentrations of desired RNA molecules such as
enzymatic nucleic
acids in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra;
Noonberg et al., 1994,
Nucleic Acid Res., 22, 2830; Noonberg et al., US Patent No. 5,624,803; Good et
al., 1997, Gene
Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736;
all of these
publications are incorporated by reference herein. The above enzymatic nucleic
acid transcription
units can be incorporated into a variety of vectors for introduction into
mammalian cells, including
but not restricted to, plasmid DNA vectors, viral DNA vectors (such as
adenovirus or adeno-
associated virus vectors), or viral RNA vectors (such as retroviral or
alphavirus vectors) (for a
review see Couture and Stinchcomb, 1996, supra).
In yet another aspect the invention features an expression vector comprising
nucleic acid
sequence encoding at least one of the nucleic acid molecules of the invention,
in a manner which
allows expression of that nucleic acid molecule. The expression vector
comprises in one
embodiment; a) a transcription initiation region; b) a transcription
termination region; c) a nucleic
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
acid sequence encoding at least one said nucleic acid molecule; and wherein
said sequence is
operably linked to said initiation region and said termination region, in a
manner which allows
expression and/or delivery of said nucleic acid molecule. In another preferred
embodiment the
expression vector comprises: a) a transcription initiation region; b) a
transcription termination
5 region; c) an open reading frame; d) a nucleic acid sequence encoding at
least one said nucleic acid
molecule, wherein said sequence is operably linked to the 3'-end of said open
reading frame; and
wherein said sequence is operably linked to said initiation region, said open
reading frame and said
termination region, in a manner which allows expression and/or delivery of
said nucleic acid
molecule. In yet another embodiment the expression vector comprises: a) a
transcription initiation
10 region; b) a transcription termination region; c) an intron; d) a nucleic
acid sequence encoding at
least one said nucleic acid molecule; and wherein said sequence is operably
linked to said initiation
region, said intron and said termination region, in a manner which allows
expression and/or delivery
of said nucleic acid molecule. In another embodiment, the expression vector
comprises: a) a
transcription initiation region; b) a transcription termination region; c) an
intron; d) an open reading
15 frame; e) a nucleic acid sequence encoding at least one said nucleic acid
molecule, wherein said
sequence is operably linked to the 3'-end of said open reading frame; and
wherein said sequence is
operably linked to said initiation region, said intron, said open reading
frame and said termination
region, in a manner which allows expression and/or delivery of said nucleic
acid molecule.
Alternatively, the enzymatic nucleic acid molecules of the instant invention
can be expressed
20 from single stranded DNA expression vectors.
The invention also features a method for enhancing the effect of the nucleic
acid catalyst of the
instant invention in vivo. The method includes the step of causing the nucleic
acid catalyst to be
localized in vivo with its target. In a related aspect, the invention features
nucleic acid catalysts
which are adapted for localization with the viral target of the agent in vivo.
25 Those in the art will recognize that many methods can be used for
modification of nucleic acid
catalyst such that they are caused to be localized in an appropriate
compartment with a target.
Examples of these methods follow but are not limiting in the invention. Thus,
for example, the
nucleic acid catalysts of the invention can be synthesized in vivo from
vectors (or formed in vitro)
such that they are covalently or noncovalently bonded with a targeting agent,
examples of which are
30 well known in the art (Sullenger et al., US Patent No. 5,854,038;
Castanotto et al., Methods
Enzymol 2000;313:401-20; Rossi et al., Science 1999, 285,1685). These
targeting agents are
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
36
termed "localization signals". In addition, nucleic acid catalysts can be
synthesized in vitro and
administered in any one of many standard methods to cause the nucleic acid
catalysts to be targeted
to an appropriate cellular compartment within a patient.
By "enhancing" the effect of a nucleic acid catalysts in vivo is meant that a
localization signal
targets that nucleic acid catalysts to a specific site within a cell and
thereby causes that nucleic acid
catalysts to act more efficiently. Thus, a lower concentration of nucleic acid
catalysts administered
to a cell in vivo can have an equal effect to a larger concentration of non-
localized nucleic acid
catalysts. Such increased efficiency of the targeted or localized nucleic acid
catalysts can be
measured by any standard procedure well-known to those of ordinary skill in
the art. In general, the
effect of the nucleic acid catalyst is enhanced by placing the nucleic acid
catalyst in a closer
proximity with the target, so that it may have its desired effect on that
target. This may be achieved
by causing the nucleic acid catalysts to be located in a small defined
compartment with the target
(e.g., within a viral particle), or to be located in the same space within a
compartment, e.g., in a
nucleus at the location of synthesis of the target.
Localization signals include any proteinaceous or nucleic acid component which
naturally
becomes localized in the desired compartment, for example, a viral packaging
signal, or its
equivalent. Localization signals can be identified by those in the art as
those signals which cause
the nucleic acid catalysts to which they are associated with to become
localized in certain
compartments, and can be readily discovered using standard methodology
(Sullenger et al., US
Patent No. 5,854,038; Shaji et al., US Patent No. 5,834,186). These
localization signals can be
tethered to the nucleic acid catalysts by any desired procedure, for example,
by construction of a
DNA template which produces both the localization signal and nucleic acid
catalysts as part of the
same molecule, or by covalent or ionic bond formation between two moieties.
All that is essential
in the invention is that the nucleic acid catalysts be able to have its
inhibitory effect when localized
in the target site, and that the localization signal be able to localize that
nucleic acid catalysts to that
target site. For example, localization signals such as HIV's Rev response
element can be linked to
the DNA enzyme to sort in some unique way. Those skilled in the art will
recognize that other
nucleic acid localization elements may be attached to the DNAzymes of the
instant invention using
methods known in the art.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
37
Other examples include any cellular RNA/DNA localization signal which causes
RNA/DNA
containing the signal to be sorted into a pathway which does not contain large
numbers of incorrect
targets; viral protein localization/assembly signals, e.g., Rev or gag
proteins.
Increasing the concentration of a viral inhibitor at an intracellular site
important for viral
replication or assembly is a general way to increase the effectiveness of
nucleic acid catalysts. The
above-described co-localization strategy can make use of, for example, a viral
packaging signal to
co-localize nucleic acid catalysts with a target responsible for viral
replication. In this way viral
replication can be reduced or prevented. This method can be employed to
enhance the effectiveness
of nucleic acid catalysts by tethering them to an appropriate localization
signal to sort them to the
therapeutically important intracellular and viral location where the viral
replication machinery is
active.
Such co-localization or regulation of enzymatic nucleic acid molecule
strategies are not
limited to using naturally occurnng localization and regulation signals.
Nucleic acid catalysts can be
targeted to important intracellular locations by use of artificially evolved
DNA/RNAs and/or protein
decoys (Szostak, 17 TIBS 89, 1992). These evolved molecules are selected, for
example, to bind to
a viral protein and can be used to co-localize nucleic acid catalysts with a
viral target by tethering
the inhibitor to such a decoy.
By "consists essentially of ' is meant that the active enzymatic nucleic acid
molecule contains
an enzymatic center or core equivalent to those in the examples, and binding
arms able to bind
target nucleic acid molecules such that cleavage at the target site occurs.
Other sequences can be
present which do not interfere with such cleavage.
The nucleic acid catalyst of the instant invention can be used to inhibit
expression of foreign
or endogenous genes, in vitro or in vivo, in prokaryotic cells or in
eukaryotic cells, in bacteria, fungi,
mycoplasma, archebacteria, algae, plants or any other biological system.
By "endogenous" gene is meant a gene normally found in a cell in its natural
location in the
genome.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
38
By "foreign" or "heterologous" gene is meant a gene not normally found in the
host cell, but
that is introduced by standard gene transfer techniques or acquired as result
of an infection (e.g.,
bacterial, viral or fungal infection).
By a "plant" is meant a photosynthetic organism, either eukaryotic and
prokaryotic.
Examples
The following are non-limiting examples showing the selection, isolation,
synthesis and
activity of enzymatic nucleic acids of the instant invention.
Applicant employed in vitro selection to isolate populations of Mgz+-dependent
self cleaving
DNA enzymes from a pool of random-sequence molecules. Characterization of a
small number of
individual DNAzymes from various populations revealed the emergence of at
least six classes of
DNA enzymes that adopt distinct secondary structure motifs. None of the six
classes corresponds to
a previously known folding pattern. Each prototypic DNA enzyme promotes self
cleavage with a
chemical rate enhancement of at least 1000-fold above the corresponding
uncatalyzed rate.
Example 1: In vitro selection of catalytic DNAs from a random pool
The initial random population was created by ligating a pool of molecules, R19
(5'-
ACGTGTGCAGCTTTC-3') (SEQ ID NO 111) connected by a random region [N50]
followed by
(5'-TTATTACGGTAACGTTGGCAC-3') (SEQ ID NO 112), where N50 indicates the random
region of 50 nucleotides) to a synthetic RNA/DNA chimeric substrate, 2.26 (5'-
GGCACACCACAAGAGUAAUAAUGAAAGAAGCGACGCT-3') (SEQ ID NO 113), where
underlined type indicates the RNA region) using T4 DNA ligase and the oligo
1.30 (5'-
TGCACACGTAGCGTCGCT-3') (SEQ ID NO 114) to template the ligation. The pool of
synthetic
R19 molecules had first been phosphorylated and end-labeled with (y-3zP]ATP
using T4
polynucleotide kinase to provide the S' phosphate necessary for T4 ligase. The
full-length population
was separated from unligated components by denaturing PAGE. Following elution
from the gel, the
full-length molecules were ethanol precipitated and resuspended either in
0.001% SDS (early
rounds) or directly in 1X selection buffer (later rounds). The population was
then reacted at 37°C in
the selection buffer and successful catalysts were separated from unreacted
molecules by denaturing
PAGE. A zone of gel was excised to encompass all possible RNA cleavage events.
Eluted catalysts
were amplified by PCR using Taq DNA polymerase and primers 1.1 (5'-
GTGCCAACGTTACCG-
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
39
3') (SEQ ID NO 115) and 2.29 (5'-ACGTGTGCAGCTTC-3') (SEQ ID NO 116) to create
multiple
copies of the successful catalysts. A portion of the reaction was then
amplified with PCR using
primers 1.1r (5'-GTGCCAACGTTACCG-3', which terminates with a ribose) (SEQ ID
NO 117) and
2.29 to produce molecules with a ribose imbedded in the negative strand. The
negative strand was
cleaved at this ribose with alkali and heat. The positive strand was then
separated from the negative
strand fragments by denaturing PAGE. The positive strand was phosphorylated
and ligated to the
substrate 2.26 as for the initial population. The new pool was then purified
from un-incorporated
substrate and ligation template by denaturing PAGE for the next round of
selection. Figure 4 is a
schematic representation of the selection process described above.
Generation 0 composed of the initial random pool was reacted at 37°C
for 12 hours in a buffer
that was 50 mM magnesium chloride, 20 mM for amino acids with pKa's nearest
neutral (arg, asn,
cys, gln, glu, his, lys, met, phe, ser), 1 mM for tyrosine (due to its low
solubility), and 2 mM for the
remaining natural amino acids. The selection buffer also contained, in every
round, 150 mM
potassium chloride, 7 mM putrescine (to potentially assist in folding), and 10
mM reduced
glutathione and was adjusted to pH 7.6 ~ 0.1 at 37°C after addition of
all components. Both the
reaction time and the concentrations of magnesium and the amino acids were
reduced over the
course of selection to final values of 10 minutes reaction time and 0.5 mM of
both magnesium and
each amino acid. When the amino acid content was reduced, HEPES was added at
50 mM to
compensate for the loss in buffering.
During the selection process, the times allowed for ligation and for elution
of the pool from
the ligation acrylamide gel were reduced to prevent RNA cleavage during these
processes.
Glutathione was included in the selection buffer at a cytoplasm-like
concentration to keep the
components, especially cysteine, reduced. As an additional precaution, the
cysteine and glutathione
were resuspended and added fresh to the selection buffer just before use
(early rounds) or the freshly
prepared solution was frozen in single-use aliquots and thawed just before use
(later rounds).
Additionally, arginine was included in the cysteine/glutathione mix because
arginine is also
somewhat volatile in that its solutions absorb cardon dioxide from air, thus
potentially changing the
pH.
In vitro selection was performed for the purpose of discovering novel
deoxyribozymes ideally
suited for cleaving RNA in cells. Several new classes of magnesium dependent
RNA-cleaving DNA
sequences have been recovered. These enzymes show no primary sequence
similarity to known
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
deoxyribozymes performing the same chemical reaction. Prototypic unimolecular
representatives
from each of these classes self cleave at imbedded RNA moieties with observed
rate constants of
10-Z miri' in 0.5 mM MgCl2, with 150 mM KCI, 7 mM putrescine, 10 mM reduced
glutathione, and
SO mM HEPES pH 7.6 at 37°C. A shortened version of the sequence of the
dominant class, class I,
5 has a catalytic core of 20 nucleotides. This deoxyribozyme can be made to
cleave the target in trans
with a pseudo first order rate constant of at least 0.06 miri', can be
generalized to cleave an in vitro
transcribed RNA target of a different sequence, and has a pKa for a
catalytically critical functional
group of ~7.9. Its kinetic rate increases in higher concentrations of
magnesium but its maximum rate
has yet to be determined.
10 The construct on which the library was based is shown in Figure 1 and the
selection scheme
utilized is shown in Figure 4. Capitol letters indicate the four standard DNA
bases and RNA bases
(reverse type) in regions of the molecular library which were constant among
all molecules. These
regions provided the RNA substrate, positioned the random region adjacent to
the substrate by base-
pairing and allowed manipulation of the library during selection using
standard methods. Catalytic
15 sequences were derived from the region of 50 nucleotides, imbedded between
the constant regions,
synthesized to contain random DNA sequences.
Molecules were selected on the basis of their ability to self cleave at an RNA
residue.
Molecules were reacted in solution in a buffer which approximated the
composition of Escherichia
coli cytoplasm with respect to conditions expected to be relevant to RNA
cleavage (Figure 1,
20 legend). Incubations were performed at 37°C. Magnesium ions and
amino acids were initially
included in the selection buffer at concentrations well above cytoplasmic
levels in order to favor
RNA cleavage so as to increase the copy number of deoxyribozymes from the
random library.
Active catalysts were isolated from the remainder of the pool by size
separation for all possible
cleavage events within the RNA region with denaturing PAGE. Once a catalytic
population had
25 been established, the conditions of the selection were made increasingly
more stringent by
decreasing the time the molecules were allowed to react and by decreasing the
concentration of all
buffer components to physiologic levels.
No known DNAzyme motif has been identified amongst the six classes of DNAs
examined
from the current selection. Instead entirely new catalytic motifs have been
discovered (Table 2) that
30 have no detectable primary or secondary sequence similarity to known RNA-
cleaving
deoxyribozymes. Representative clones from each class were tested in their
unimolecular format to
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
41
determine catalytic rate and cleavage site (Table 3). These new sequences may
represent unique
tertiary structures having altered kinetic properties. Studies are in progress
to assess each class
individually.
Clone 27 of class I was studied in a minimized, bimolecular format (Figure 2).
The secondary
structure shown in Figure 2a is based on the region of conserved nucleotides
underlined in Table 2
with base-pairing restored at the 3' end of the catalyst. The Kd for this
enzyme interacting with
substrate is 5 ~M, which may reflect poor binding due to the A-U rich nature
of the putative
substrate binding arms. The enzyme does not require glutathione but does lose
10-fold activity in the
absence of putrescine. Its pKa is ~ 7.9, reaching a maximum pseudo first order
rate constant in 0.5
mM Mg2+ of 0.06 miri'. The enzyme also cleaves the in vitro transcribed target
shown in Figure 2b,
demonstrating that it does not require DNA residues in the substrate and can
be generalized by
changing the substrate binding arms. The relative cleavage rates of the Class
I motif on various
substrates shown in Figure 6 are presented in Table 4. Under single-turnover
conditions, the rate of
each enzyme is limited by binding a single magnesium ion with a Kd of 1 mM
(Figure 6).
Therefore, the catalytic core is well adapted to the magnesium and pH levels
in cells. The pseudo
first order rate constant for RNA cleavage under simulated physiologic
conditions (pH 7.6, 2 mM
Mgz+, 150 mM K+, 7 mM putrescine, 37°C) is 0.060 miri'. The maximum
rate constant at room
temperature with MgZ+ increased to 8 mM and pH at 8.9 is 0.22 miri'.
The Class I motif has been targeted to cleave a variety of RNA substrates
under simulated
physiologic, single-turnover conditions by altering the putative substrate
binding regions to be
complementary to new targets. Additional cleavage sites are shown in Figure 5.
The cleavage rates
relative to the model substrate from Figure 2 are summarized in Table 4 and
show wide variation.
This variation is not due to an inability to recognize an all-RNA substrate
over an RNA/DNA
chimera, as was present in the original selection but is most likely a result
of intramolecular structure
of the RNA targets. The substrate in Figure SA corresponds to a portion of the
5' UTR and ORF of
an mRNA of a bacterial protein while the substrate in Figure SB was
specifically designed for this
test. The suitability of the latter substrate was checked by the secondary
structure prediction program
mfold (Mathews et al., 1999, J. Mol. Biol., 288, 911-940) version 3.1 such
that: (1) the substrate was
predicted not to have significant intramolecular structure; (2) each
deoxyribozyme would interact
with only one site on the RNA; (3) and each deoxyribozyme/substrate base-
pairing interaction
would 4 to 8 kcal/mole more stable than the model substrate interaction
indicated in Figure 2.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
42
Nevertheless, tertiary and non-standard interactions that may have significant
effects, especially
intramolecularly, may not be predicted.
Class IV and class V motifs can also cleave substrates in trans (Figure 7).
The class IV clone
sequences from in vitro selection contained an extended stem-loop in the core
that has been
truncated, making the core size no more than 29 nucleotides. Likewise, class V
has been truncated to
a core size of 28 nucleotides. The Class IV motif can be targeted to cleave an
in vitro transcribed
RNA with a pseudo first order rate constant of 0.002 min-1 in 0.5 mM Mg2+, 150
mM K+, 7 mM
putrescine, pH 7.6, 37°C. With this substrate, the Kd is approximately
1 pM in the same buffer. The
motif has been shown to have no requirement for glutathione. The rate constant
of the class V motif
is estimated to be approximately 0.006 min-1 when tested in a similar buffer
as above but with 10
mM glutathione included.
Studies were performed on clones 27 and 37 of class II. As for clone 27, an
attempt was made
to synthesize a molecule thought to be the minimal catalytically active
sequence but with perfect
substrate recognition sites (Figure 3). There is a dramatic loss of activity
with this construct. An
unligated bimolecular assay on the original clone 37 where the entire sequence
of the full-length
molecule was present but the molecule was severed in the connecting loop
region was also inactive.
The other motifs have been studied unimolecularly, cleaving RNA in cis. The
class III clone,
21, does cleave in an unligated bimolecular assay. However, the poor
procession of the unimolecular
reaction (only 7% processed, Table 3) indicates an interference of some sort,
possibly one that
might be solved by truncation if more structural data were available. Clone 34
does show activity
but apparently activates during elution from the purification gel following
preparation. Clone 34
reactions show significant cleavage at time zero but no additional cleavage
under several buffer
conditions, including those designed to mimic the elution buffer. An isolated
class VI motif has
been shown to be a magnesium dependent catalyst with an estimated cleavage
rate constant of 0.002
min-1 (0.5 mM Mgz+, 150 mM K+, 7 mM putrescine, 10 mM glutathione pH 7.6,
37°C).
DNAzyme Engineering
Sequence, chemical and structural variants of DNAzymes of the present
invention can be
engineered using the techniques shown above and known in the art to cleave a
separate target RNA
or DNA in traps. DNAzymes can be reduced or increased in size using techniques
known in the art.
Techniques described for engineering molecules containing 2'-hydroxyl (2'-OH)
groups can be
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
43
applied to the DNA enzymes of the instant invention (for example, see Zaug et
al., 1986, Nature,
324, 429; Ruffner et al., 1990, Biochem., 29, 10695; Beaudry et al., 1990,
Biochem., 29, 6534;
McCall et al., 1992, Proc. Natl. Acad. Sci., USA., 89, 5710; Long et al.,
1994, supra; Hendry et al.,
1994, BBA 1219, 405; Benseler et al., 1993, JACS, 115, 8483; Thompson et al.,
1996, Nucl. Acids
Res., 24, 4401;Michels et al., 1995, Biochem., 34, 2965; Been et al., 1992,
Biochem., 31, 11843;
Guo et al., 1995, EMBO. .1., 14, 368; Pan et al., 1994, Biochem., 33, 9561;
Cech, 1992, Curr. Op.
Struc. Bio., 2, 605; Sugiyama et al., 1996, FEBS Lett., 392, 215; Beigelman et
al., 1994, Bioorg.
Med. Chem., 4, 1715; all of these references are incorporated in their
totality by reference herein).
For example, the stem-loop domains of the DNAzymes may not be essential for
catalytic activity
and hence can be systematically reduced in size using a variety of methods
known in the art, to the
extent that the overall catalytic activity of the DNAzyme is not significantly
decreased. In addition,
the introduction of variant stem-loop structures via site directed mutagenesis
and/or chemical
modification can be employed to develop DNAzymes with improved catalysis,
increased stability,
or both.
Further rounds of in vitro selection strategies described herein and
variations thereof can be
readily used by a person skilled in the art to evolve additional nucleic acid
catalysts and such new
catalysts are within the scope of the instant invention. Additionally,
"Mutagenic PCR" (Cadwell
RC, Joyce GF PCR Methods Appl 1994 Jun 3:6 S136-40) can be used to further
optimize the
sequences described in Formulae I and II. In addition, the optimization of
these variant DNAzyme
constructs by modification of stem-loop structures as is known in the art can
provide for species
with improved cleavage activity.
Target sequence requirements for DNAzymes can be determined and evaluated
using methods
known in the art.
New ribozvme motifs
An extensive array of site-directed mutagenesis studies have been conducted
with the
hammerhead ribozyme to probe relationships between nucleotide sequence and
catalytic activity..
These systematic studies have made clear that most nucleotides in the
conserved core of the
hammerhead ribozyme (Forster & Symons, 1987, Cell, 49, 211) cannot be mutated
without
significant loss of catalytic activity. In contrast, a combinatorial strategy
that simultaneously screens
a large pool of mutagenized ribozymes for RNAs that retain catalytic activity
can be used more
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
44
efficiently to define immutable sequences and to identify new ribozyme
variants (Breaker, 1997,
supra). For example, Joseph and Burke (1993; J. Biol. Chem., 268, 24515) have
used an in vitro
selection approach to rapidly screen for sequence variants of the 'hairpin'
self cleaving RNA that
show improved catalytic activity. This approach was successful in identifying
two mutations in the
hairpin ribozyme that together give a 10-fold improvement in catalytic rate.
Applicant employed in vitro selection to isolate populations of Mg2+-dependent
self cleaving
ribozymes from a pool of random-sequence molecules. Characterization of a
small number of
individual ribozymes from various populations revealed the emergence of at
least 12 classes of
ribozymes that adopt distinct secondary structure motifs. Only one of the 12
classes corresponds to
a previously known folding pattern - that of the natural hammerhead ribozyme.
Each prototypic
ribozyme promotes self cleavage via an internal phosphoester transfer reaction
involving the
adjacent 2'-hydroxyl group with a chemical rate enhancement of at least 1000-
fold above the
con-esponding uncatalyzed rate.
In vitro selection of self cleaving RNAs from a random pool
Isolation of new ribozymes from a population of 10'4 different RNAs was
performed using
the selective-amplification scheme depicted in Figure 8A. Each RNA construct
used for in vitro
selection includes a 40-nt random-sequence region flanked both on its S'- and
3' sides by domains
of defined nucleotide sequence (Figure 8B). The 3'-flanking domain was
designed to serve as the
target site for ribozyme cleavage. Due to its extended length, the 3'-flanking
domain can experience
the loss of nucleotides via ribozyme action, yet still function as a primer-
binding site for
amplification by RT-PCR. Moreover, the nucleotide sequence of this domain was
designed to
represent all 16 possible nearest neighbor combinations in order to favor the
isolation of ribozymes
that might have necessity for a particular dinucleotide identity at their
cleavage site.
In each round of selection, the RNAs were transcribed in vitro using T7 RNAP
and the
uncleaved RNA precursors were isolated by PAGE. The Mg2+ concentration and the
incubation time
used for in vitro transcription were minimized in order to reduce the
likelihood that efficient Mg2+-
dependent ribozymes would be cleaved during enzymatic synthesis and thereby
lost upon isolation
of the uncleaved RNA precursors. In addition, the incubation time used for
crush/soak isolation of
RNA precursors from polyacrylamide gel was minimized to preclude the isolation
of self cleaving
ribozymes that react in the absence of Mg2+. For selection, the purified RNA
precursors were
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
incubated under permissive reaction conditions (50 mM Tris-HC1 [pH 7.5 at
23°C], 250 mM KC1,
20 mM MgClz) at 23°C for 4 hr. RNAs that cleaved in the 3' domain
during this incubation were
isolated by PAGE. Specifically, the zone that contains RNA cleavage fragments
that are between
~10 to ~30 nucleotides shorter than the precursor RNAs was excised and the
recovered RNAs were
5 amplified by RT-PCR.
The population of RNAs isolated after six rounds of selection (G6) exhibits a
significant level
of self cleavage activity in the presence of Mg2+. At this stage, the major 5'-
cleavage products were
isolated and the RT-PCR products of the isolated RNAs were cloned and
sequenced. The remainder
of the zone typically excised, which presumably contains 5'-cleavage fragments
of different lengths,
10 was used to continue the selective-amplification process. This isolation
strategy serves two
purposes. First, the ribozymes that produce the major cleavage products
observed in Fig. 1 C, and
which presumably dominate the population at G6, can be examined in greater
detail. Second, these
more commonly represented sequences are largely excluded from the subsequent
rounds of selection
and thus are less likely to dominate the RNA population in future rounds. This
allows ribozymes
15 that are less frequently represented to increase in frequency in later
populations. In a similar fashion,
the main product bands representing the 5'-cleavage fragments at G9, G12 and
G15 were selectively
recovered from the gel and subjected to RT-PCR amplification followed by
cloning and sequencing.
This strategy offers an effective means by which many different self cleaving
RNA motifs can be
isolated.
20 By "random sequence region" is meant a region of completely random sequence
and/or
partially random sequence. By completely random sequence is meant a sequence
wherein
theoretically there is equal representation of A, T, G and C nucleotides or
modified derivatives
thereof, at each position in the sequence. By partially random sequence is
meant a sequence wherein
there is an unequal representation of A, T, G and C nucleotides or modified
derivatives thereof, at
25 each position in the sequence. A partially random sequence can therefore
have one or more
positions of complete randomness and one or more positions with defined
nucleotides.
Characterization of New self cleavin, riboz, m~quences
Sequence analysis of greater than 100 clones representing self cleaving
ribozymes from the
G6, G9, G 12 and G1 S populations revealed as many as 20 distinct sequence
classes of RNAs. In
30 some cases, numerous RNAs conformed to a single class of self cleaving
ribozyme based on
sequence elements that are common among the related variants. For example, ~30
clones were
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
46
identified that can form a common secondary structure with similar core
nucleotides that we defined
as "class I" ribozymes. In other instances, RNAs were isolated that were
entirely unique in sequence
compared to all other clones. These "orphan" sequences were classified
independently, for example,
as was done with the "class II" ribozyme. Ultimately, these 20 different
sequence classes were
grouped into twelve distinct structural classes of self cleaving ribozymes
that were identified based
on the presence of distinctive sequence elements, secondary structures and
cleavage sites (Figure 9).
Only one of the twelve classes (class IX) corresponds to a naturally-occurring
RNA - the self
cleaving hammerhead ribozyme. The distinctions between the different
structural classes of
ribozymes were established as described for class I and class II ribozymes
below.
For each of the 20 putative sequence classes into which the ribozymes were
originally
grouped, applicant conducted three to five rounds of reselection (Figure 8A)
for self cleavage
activity beginning with a population of ribozyme variants based on the
nucleotide sequence of a
representative clone. As expected, the introduction of random mutations (d =
0.21; see Fedor and
Uhlenbeck, 1992, Biochemistry, 31, 12042-12054) into the original N4o domain
of each clone
decreases the catalytic activity of the population significantly. However,
after three to five rounds of
reselection, the mutagenized populations recovered significant levels of self
cleavage activity. Once
the catalytic activity of a particular population became near equal to that of
its parent clone,
representative ribozyme variants were identified by cloning and sequencing.
The nucleotide sequences corresponding to the variant ribozyme domains of
cloned RNAs for
each class were aligned to provide an artificial phylogeny. Table 11 shows
artificial phylogenetic
analysis of class I, class II, and class V ribozymes. Sequence variations are
depicted for the 40
nucleotides corresponding to the original random-sequence domain of each
parental sequence
(numbered). Dots indicate no change from the parental sequence listed first.
The asterisk identifies a
class I ribozyme variant that carries mutations at positions 18 and 26 that
retain the ability to base
pair. For example, applicant observed that the parental sequences of both
class I and class II
ribozymes undergo substantial mutation without complete disruption of
catalytic activity (Table 11).
However, the pattern of mutation acquisition in each case is indicative of the
presence of conserved
primary and secondary structures that presumably are important for ribozyme
function. Among the
ribozyme variants obtained for class I, all retain the prototypic nucleotide
sequence at positions 12-
16 (Figure 9 and Table 11), suggesting that these nucleotides are critical for
self cleavage activity.
Nucleotides 8-14, which overlap with the conserved 12-16 nt region exhibit
complementarity to
nucleotides within the 3' primer-binding domain. Moreover, the nucleotides at
positions 17-19 and
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
47
25-27 are both mutually complementary and highly conserved. This indicates
that these latter two
sequence elements also might form a hairpin structure with intervening
nucleotides 20-24 serving as
a connecting loop. Consistent with this interpretation is the observation that
mutations within
positions 17-19 and 25-27 acquired by a class I ribozyme variant (Table 11;
asterisk) allows
retention of base complementarity. In addition, the putative loop sequence
spanning nucleotides 20-
24 tolerates significant mutation as would be expected if the nucleotides in
the loop were
unimportant for stem formation and ultimately for ribozyme action.
Similar characteristics of structure formation and conserved sequences are
evident in the
artificial phylogeny for class II ribozymes (Table 11). However, the data
indicate that class II
ribozymes form a catalytic structure that is distinct from that of class I.
For example, complementary
segments of the original random-sequence domain also are consistent with the
formation of two
separate stem elements. However, if present, these stems would be arranged
differently from those
predicted to form in class I ribozymes. At this time only one stem, that
formed between nucleotides
8: 11 and 16-19, appears to be present as judged by the emergence of sequence
variants in the
artificial phylogeny of class II. Moreover, two conserved regions (nucleotides
1-7 and 22-30)
(Figure 9 and Table 11) of class II RNAs are different from the conserved
sequences found in class
I RNAs, indicating that the two classes of ribozymes are indeed distinct.
Likewise, by analysis of the artificial phylogenetic data for the remaining 18
clones, applicant
identified as many as 12 distinct classes of ribozymes (Figure 9). In many
instances, obscure
sequence and structural similarities were revealed between the different
sequence classes upon
examination of each artificial phylogeny that was generated by reselection.
Such unforeseen
similarities were identified typically among representative RNAs that conform
to a structural class
placing very little demand on sequence conservation in the core of the
ribozyme domain (e.g. classes
V and VIII). It also is important to note that the structural models depicted
in Figure 9 are not
intended to represent confirmed secondary structures. In most cases, limited
artificial phylogenetic
data coupled with preliminary analyses using a secondary-structure prediction
algorithm (Zuker,
1989, Science, 244, 48-52) were used to derive the models.
Characterization of Bimolecular Ribozvme Reactions
Based on preliminary sequence analysis, class I self cleaving ribozymes form
an X-shaped
secondary structure (Figure 9). In this model, a separate ribozyme domain base
pairs to its
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
48
corresponding substrate domain with duplex formation occurring both 5' and 3'
relative to an
unpaired G residue that resides within the nearest neighbor sequence of the
original RNA construct
(position 4 of the nearest neighbor domain, Figure 8B; position 9 of a 21-nt
substrate "S21", Figure
9). This secondary structure arrangement also locates the conserved
nucleotides and the putative
hairpin structures near this unpaired G residue.
Phosphodiester linkages of residues that reside outside of helical structures
are most likely to
be targets for ribozyme action. While stable RNA helices restrict
conformational freedom of
phosphodiester bonds, linkages joining unpaired nucleotides can more easily
adopt the "in-line"
geometry that is necessary for internal transesterification (Soukup, 1999,
RNA, 5, 1308-1325).
Therefore, the structural model indicates that cleavage of S21 should occur at
the G residue residing
at position 9 (Figure 9). Consistent with the proposed "X-motif ' model is the
observation that a
bimolecular construct comprised of S21 and a 46-nt RNA corresponding to the
enzyme domain of a
minimized X motif variant (Figure 9) exhibits Mgz+-dependent cleavage
activity. Furthermore, the
RNA cleavage was determined to occur at the phosphodiester linkage that
resides 3' relative to the
unpaired G (between nucleotides 9 and 10) of 521.
Applicant also examined the cleavage activity of a 44-nt construct that carnes
the conserved
sequence and structural elements identified in class II ribozymes. This RNA
forms a bimolecular
interaction with S21 that presumably leaves unpaired the substrate nucleotides
G and A at positions
6 and 7 (Table 11). Consistent with this structural model, we find that the
complex is cleaved within
S21 between nucleotides 6 and 7 when MgZ+ is included in the reaction mixture.
In a similar
fashion, bimolecular structures were tested for eight of the remaining 10
classes. In most instances,
ribozyme activity and cleavage patterns are consistent with the proposed
secondary structures
depicted in Figure 9.
Rate Constants for Prototypic Constructs Representing the 12 Ribozyme Classes
Rate constants for RNA transesterification were determined for each of the 12
constructs
depicted in Figure 9. For example, both class I and class II constructs
produce linear cleavage
kinetics through more than one half life of the substrate RNA (Figure 10A).
The representative class
I and class II constructs examined herein exhibit rate constants of 0.01 and
0.05 miri', respectively.
Similar examinations of the remaining ribozyme constructs reveal that the rate
constants for RNA
cleavage in nearly all cases are below 0.05 miri'. In contrast, rate constants
for the natural self
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
49
cleaving RNAs such as the hammerhead and HDV ribozymes typically range between
1 miri' and
100 miri' (Fedor and Uhlenbeck, 1999, Biochemistry, 31, 12042-12054). For
comparison, the
uncatalyzed rate of internal RNA transesterification under the selection
conditions used is ~10-' miri
' (Li and Breaker, 1999, J. Am. Chem. Soc., 121, 5364-5372). Therefore, even
the slowest of
constructs depicted in Figure 9 accelerate the chemical step of RNA cleavage
by at least 1,000 fold.
The most active prototype construct (class VIII, kobs - 0.1 miri') provides an
overall chemical rate
enhancement of ~1 million fold over the corresponding uncatalyzed rate.
It is important to note, however, that the original selection and the 20
independent reselections
were conducted such that even very slow ribozymes were allowed to persist in
the population. The
isolation of ribozymes from random sequence employed ribozyme incubation times
of 4 hr. Under
these selection conditions, RNAs that self cleave with a rate constant greater
than 10-3 miri'
experience little or no selective disadvantage compared to ribozymes that
cleave with infinitely
faster cleavage rates. Similarly, the goal of the reselections was to create
variant ribozymes that
retained activity so that an artificial phylogeny could be created for each
representative RNA. The
ribozyme incubations during reselection were typically carried out for 30-60
min. Therefore, the
selective pressure applied at this stage also was not sufficient to favor the
isolation of ribozymes
with rates that rival those of naturally occurnng ribozymes.
Ribozyme Cleava a
Ribozymes and deoxyribozymes that catalyze the cleavage of RNA via a cyclizing
mechanism
typically require a specific consensus sequence at the site of cleavage. In
most cases, substrate
binding specificity is determined by Watson/Crick base pairing between enzyme
and substrate
domains, and this specificity can easily be engineered by the user. However,
the hammerhead
ribozyme favors cleavage of the phosphodiester linkage at UH sites, where H
represents A, U or C
(Vaish et al., 1998, PNAS USA, 95, 2158-2162). To maximize the diversity of
ribozymes that could
be isolated by applicant's selection scheme, applicant included the nearest
neighbor domain depicted
in Figure 8B. This domain provides a comprehensive sampling of all 16
dinucleotides, thereby
offering a greater diversity of cleavage sites than would be present in most
substrate domains of
arbitrary sequence composition. Applicant found that the 12 classes of
ribozymes examined in this
study cleave at five different locations within the nearest neighbor domain
(Figure 10B). As
predicted from RNA cleavage patterns, ribozymes isolated from the G6
population (classes I-IV)
cleave at two sites - both near the 5' end of this domain. Similar
correspondence between cleavage
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
patterns and cleavage site selection are observed with ribozymes isolated from
subsequent rounds.
However, ribozymes from G12 and G15 that generate the largest 5'-cleavage
fragments are not
representative of new cleavage sites near the 3' end of the nearest neighbor
domain. Applicant fords
that several ribozymes accumulated insertions in the original NQ°
domain that yield longer 5'-
5 cleavage fragments despite processing at sites that are also used by other
ribozymes. The most
frequently used cleavage site is the linkage between the initial GA
dinucleotide at the 5' end of the
nearest neighbor domain, (Figure 10B). However, it is not clear whether or not
this dinucleotide
sequence is intrinsically favorable to the formation of structures that self
cleave.
Class I "X motif'
10 Nearly all ribozyme constructs derived from the reselections (Figure 9)
exhibit rate constants
for RNA cleavage that are far below the maximum values obtained for the
natural self cleaving
ribozymes. This observation brings into question whether new RNA-cleaving
motifs can even
approach the catalytic efficiency of motifs that currently exist in nature.
Until this point, applicant's
in vitro selection and reselection efforts have been directed towards the
identification and
15 confirmation of new structural motifs for self cleaving ribozymes.
Therefore, the selection reactions
intentionally were conducted under exceedingly permissive conditions such that
even highly
defective variants of new motifs could be enriched in the RNA population and
ultimately be
identified by cloning and sequencing. Likewise, reselections were conducted
under almost equally
permissive conditions in order to identify variants that could contribute to
the assembly of artificial
20 phylogenies, and not necessarily to produce superior catalysts. Indeed,
none of the 20 reselections
conducted, including one that was carried out using a mutagenized population
derived from
hammerhead i, provided variant ribozymes that exhibit kinetic characteristics
like those displayed by
natural self cleaving ribozymes.
Therefore applicant set out to enrich for ribozyme variants whose rate
constants were
25 significantly higher than that of the general population. To this end,
applicant pooled RNA samples
from G6, G9, G12 and G15. This combined starting population was then subjected
to additional
rounds of in vitro selection as described in Figure 8A, but where decreasing
incubation times (as
low as 5 sec) for the selection reaction were used to favor the isolation of
the fastest ribozymes.
After 10 additional rounds of selection, applicant found that the resulting
RNA population
30 undergoes ~6% cleavage upon incubation for S sec under the permissive
reaction conditions.
Surprisingly, only a single 5'-cleavage fragment is observed despite the fact
that ribozymes cleaving
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
51
at multiple sites within the nearest neighbor domain were selectively
amplified. These results
indicated that a single class of relatively fast ribozymes (kobs ~0.7 miri')
has come to dominate the
RNA population. Indeed, cloning and sequencing revealed that the population is
comprised
primarily of a single RNA sequence (Figure 11B) that corresponds to a
variation of the original X
motif ribozyme (Figure 9, class I).
A bimolecular construct based on the dominant self cleaving RNA (Figure 11B)
exhibits a kobs
of ~0.2 miri' under single turn-over conditions, which corresponds to a 20-
fold improvement over
the corresponding class I construct depicted in Figure 9. This finding
suggests that further
improvements in catalytic performance might be made to the remaining classes
of ribozymes, as the
initial constructs examined likely reflect non-optimal variants.
Interestingly, the maximum rate
constant expected for optimal hammerhead ribozymes (~1 miri') remains several
fold greater than
that measured for the bimolecular version of the improved X motif depicted in
Figure 11B.
Nevertheless, applicant concludes from these findings that other small RNA
motifs can form RNA-
cleaving structures that compare favorably in catalytic rate enhancement with
that of natural
ribozymes.
Ribozyme Engineering
Sequence, chemical and structural variants of ribozymes of the present
invention can be
engineered using the techniques shown above and known in the art to cleave a
separate target RNA
or DNA in traps. For example, the size of ribozymes can be reduced or
increased using techniques
known in the art (Zaug et al., 1986, Nature, 324, 429; Ruffner et al., 1990,
Biochem., 29, 10695;
Beaudry et al., 1990, Biochem., 29, 6534; McCall et al., 1992, Proc. Natl.
Acad. Sci., USA., 89,
5710; Long et al., 1994, Supra; Hendry et al., 1994, BBA 1219, 405; Benseler
et al., 1993, JACS,
115, 8483; Thompson et al., 1996, Nucl. Acids Res., 24, 4401;Michels et al.,
1995, Biochem., 34,
2965; Been et al., 1992, Biochem., 31, 11843; Guo et al., 1995, EMBO. J., 14,
368; Pan et al., 1994,
Biochem., 33, 9561; Cech, 1992, Curr. Op. Struc. Bio., 2, 605; Sugiyama et
al., 1996, FEBSLett.,
392, 21 S; Beigelman et al., 1994, Bioorg. Med. Chem., 4, 1715; all are
incorporated in their totality
by reference herein). For example, the stem-loop domains of the ribozymes may
not be essential for
catalytic activity and hence could be systematically reduced in size using a
variety of methods
known in the art, to the extent that the overall catalytic activity of the
ribozyme is not significantly
decreased. In addition, the introduction of variant stem-loop structures via
site directed mutagenesis
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
52
and/or chemical modification may be employed to develop ribozymes with
improved catalysis,
increased stability, or both.
Further rounds of in vitro selection strategies described herein and
variations thereof can be
readily used by a person skilled in the art to evolve additional nucleic acid
catalysts and such new
catalysts are within the scope of the instant invention. In addition, the
optimization of these variant
ribozyme constructs by modification of stem-loop structures as is known in the
art may provide for
species with improved cleavage activity.
Example 2: Oligonucleotide synthesis
Synthetic DNAs and the 21 nucleotide (nt) RNA substrate (S21) were prepared by
standard
solid phase methods (Keck Biotechnology Resource Laboratory, Yale University)
and purified by
denaturing (8 M urea) polyacrylamide gel electrophoresis (PAGE). The 2'-TBDMS
groups of the
synthetic RNA were removed by 24 hr treatment with triethylamine
trihydrofluoride (15 p1 per
AU26° crude RNA). Molecules that were radiolabeled using T4
polynucleotide kinase (T4 PNK, New
England Biolabs) and [y-32P]ATP according to the manufacturer's directions
were subsequently
purified by denaturing 20% PAGE and isolated from the gel by crush-soaking in
10 mM Tris-HCl
(pH 7.5 at 23°C), 200 mM NaCI and 1 mM ethylenediaminetetraacetic acid
(EDTA) followed by
precipitation with ethanol as described below.
Example 3: In vitro Selection
The initial population of RNA for in vitro selection was created by first
generating a double-
stranded DNA template for in vitro transcription. SuperScript~ II reverse
transcriptase (RT,
GibcoBRL) was used to extend 280 pmoles of the DNA oligonucleotide "primer 1"
(5'-
GAAATAAACTCGCTTGGAGTAACCATCAGGAC-AGCGACCGTA-3') (SEQ ID NO 118);
region representing 16 possible nearest neighbor combinations is underlined)
using 270 pmoles of
the template DNA (5'- TCTAATACGACTCACTATAGGAAGACGTAGCCAA-3') (SEQ ID NO
119) followed by a random region (NQ°) followed by (S'-TACGGTCGCTGTCCTG-
3') (SEQ ID NO
120); T7 promoter is underlined and N represents an equal mixture of the four
standard nucleotides).
The extension reaction was conducted in a total of 50 p1 containing 50 mM Tris-
HCl (pH 8.3 at
23°C), 75 mM KCI, 3 mM MgCl2, 10 mM dithiothreitol (DTT), 0.2 mM each
of the four
deoxyribonucleoside-5' triphosphates (dNTPs), and 10 U p1-' RT by incubation
at 37°C for 1 hr. The
resulting double-stranded DNA was precipitated by the addition of S p1 3 M
sodium acetate (pH 5.5)
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
53
and 140 p1 100% ethanol and pelleted by centrifugation. This extension
reaction provides 10'4
different template sequences.
The DNA templates were transcribed in a total volume of 100 p1 containing 50
mM Tris-HCl
(pH 7.5 at 23°C), 10 mM MgClz, 50 mM DTT, 20 mM spermidine, 2 mM each
of the four
ribonucleoside-5' triphosphates (NTPs), and 35 U p1-' T7 RNA polymerise (T7
RNAP) by
incubation at 37°C for 1 hr. [a-'zP]UTP was added to the transcription
reaction to produce internally
~ZP-labeled RNAs when necessary. The reaction was terminated by the addition
of 50 p l 40 mM
EDTA and precipitated by the addition of the appropriate amounts of sodium
acetate and ethanol.
The uncleaved precursor RNAs were isolated by denaturing 10% PAGE, recovered
from the gel as
described above and stored in deionized water (dHzO) at -20°C until
use. In subsequent rounds of
selection, double-stranded DNA from the polymerise chain reaction (PCR) was
transcribed as
described above, but for shorter periods of time (e.g. 10 min) to minimize the
loss of ribozymes that
efficiently cleave during transcription.
The initial selection reaction (GO) contained 2000 pmoles of RNA in a total of
400 p1 reaction
buffer (SO mM HEPES [pH 7.5 at 23°C], 250 mM KCl and 20 mM MgClz) and
was incubated at
23°C for 4 hr. The reaction was terminated by the addition of EDTA and
the RNA was recovered by
precipitation with ethanol. RNA cleavage products were separated by denaturing
10% PAGE,
visualized and quantified using a Molecular Dynamics PhosphorImager~, and the
gel region
corresponding to the location of the desired RNA cleavage products was
excised. The RNA was
recovered from the excised gel by crush-soak elution followed by precipitation
with ethanol. The
selected RNAs were amplified by RT-PCR as described previously (10) using
primers l and 2 (5'-
GAATTCTAATACGACTCACTATAGGAA-GACGTAGCCAA-3') (SEQ ID NO 121); T7
promoter is underlined). The resulting double-stranded DNA from each round of
in vitro selection
was used to transcribe the RNA population for the subsequent round, in which
all steps were
conducted at ~1/10'h the scale of G0. All other parameters of the selection
process were maintained
as in G0. Representative ribozymes from the populations derived from 6, 9, 12
and 15 rounds of
selection were examined by cloning (TOPO~-TA cloning kit; Invitrogen) and
sequencing
(ThermoSequenase~ kit; Amersham Pharmacia).
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
54
Example 4: Artificial Phylo~y Generation
To confirm the self cleaving activity of various clones, internally 'ZP-
labeled ribozyme
precursors were incubated under the selection reaction conditions for up to
several hours to detect
cleavage products upon separation by denaturing 10% PAGE. Artificial
phylogenies for active
clones each representing one of ~20 different sequence classes were generated
by conducting
reselections after the introduction of mutations. Reselections were conducted
by first synthesizing a
DNA construct for each individual that corresponds to the template strand for
in vitro transcription.
In each case, the nucleotides corresponding to the original random-sequence
domain were
synthesized with a degeneracy (d) of 0.21 per position, such that all possible
variants with six or
fewer mutations relative to the original sequence are represented. Typically,
three to five rounds of
selective amplification as described above (using selection reaction
incubations of 1 hr or less) were
required for each population to exhibit a level of catalytic activity that
corresponded to that of the
original clone. When this occurred, the population was cloned and sequenced.
Example 5: Characterization of Ribozyme Catalytic Function
In most cases, more detailed kinetic and structural characterizations of
ribozymes from
different structural classes were conducted with a variant from the
corresponding artificial
phylogeny that contains the mutations most commonly acquired during
reselection. Initial rate
constants for self cleavage (classes VI and VII) were determined by incubating
internally 3zP-labeled
ribozyme precursors in selection buffer for various times, separating the
products by PAGE, and
quantifying the yields by PhosphorImager~. Rate constants were derived as
previously described
(Soukup and Breaker, 1999, Proc. Natl. Acad. Sci. USA, 96, 3584-3589). Rate
constants for
bimolecular ribozyme function were established using a similar strategy,
except that the reactions
were allowed to proceed through at least two half lives of the substrate. To
achieve single turnover
conditions, trace amounts of 32P-labeled substrate were incubated with S00 nM
ribozyme.
Cleavage sites for unimolecular reactions were determined by incubating 5' 3ZP-
labeled
precursor RNA in selection buffer, separating the products by denaturing 10%
PAGE and comparing
the gel mobility of the S' cleavage fragment to that of each cleavage fragment
generated by partial
RNA digestion using RNase T1 or alkali as described previously (Soukup and
Breaker, 1999, RNA,
5, 1308-1325). Similarly, the cleavage site for each bimolecular ribozyme
reaction was established
by incubating trace amounts of S' 3ZP-labeled substrate RNA with S00 nM
ribozyme and comparing
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
the products of ribozyme cleavage, RNase T1 digestion, and alkaline digestion
using denaturing
20% PAGE.
Example 6: Chemical stabilization of ribozyme motif (Class V)
Examples of stabilized Class V ribozymes and control ribozymes useful in this
study are
5 shown in Table 10. Initially, ribozymes (Class V) were designed with varying
combinations of 2'-
O-methyl nucleotides. The ribozymes were chemically synthesized. Cleavage
reactions were
carried out with 500 nM final ribozyme concentration, single turnover kinetics
in SO mM Tris-Cl pH
7.5, 150 mM KCI, 20 mM Mg2+, 37C and trace substrate. Results are summarized
in Table 5. The
all ribo wild type V ribozyme used as a control (RPI No. 14189) resulted in a
cleavage rate (Kobs) of
10 0.044 mint . Two of the 2'-O-methyl constructs (RPI No. 14880 with 6 and 7
substituted 2'-O-Me
arms, and RPI No. 14881 with 5 and 7 substituted 2'-O-Me arms), both resulted
in improved
cleavage activity with Kobs values of 0.066 min-1. RPI construct No 14880 was
subsequently used
as a control for a study to determine the effect of 2'-O-methyl substitution
in the Class V ribozyme
motif core via a 2'-O-methyl "walk" experiment using the same cleavage
reaction conditions as
15 above. The results of this study are summarized in Figure 13 with Kobs and
Kre~ values
summarized in Table 6. The effect of various combinations of 2'-O-methyl
substitutions in the
Class V ribozyme core was investigated under the same conditions. Table 7
outlines the results of
this study in which a "seven ribose" core motif emerged with a Kobs value of
0.127 min-1. This
seven ribose motif of the Class V ribozyme (RPI No. 15705) was used as the
base motif for further
20 core stabilization studies. Additional experiments focused on stabilization
of the core pyrimidine
residues of the Class V motif. Table 8 summarizes data from an experiment
determining the
cleavage activity Class V motif ribozymes with modified core pyrimidines with
phosphorodithioate,
2'-fluoro, 2'-amino, and 2'-deoxy substitutions. This study indicates that the
C7 position (Figure
13) is the most tolerant to modifications while the U4 position is the least
tolerant to modification.
25 Further reductions in ribo core residues resulted in ribozymes with
decreased activity relative to the
"seven ribo" core version of the Class V motif, however, these further
stabilized versions retained
activity. Testing of the further reduced ribo constructs in conjunction with
additional chemical
modifications is summarized in Table 9.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
56
Example 7: Similarity between Class I and Class VIII ribozymes
Two strategies were used to examine whether the Class I and Class VIII
ribozymes share
sequence and structural similarities. First, a series of fourteen variant
constructs were generated that
examined the importance of various structural features of Class VIII
ribozymes. For example, the
5'-leader sequence of Class VIII is essential, only when the extra bulge
immediately preceding the
stem II element is present. If this bulge and putative stem II element is
replaced by a more
convincing stem II structure like that present in the Class I motif structure
(Figure 14), then the
leader sequence of Class VIII can be eliminated. In a similar fashion, other
elements of Class VIII
ribozymes when examined, show similarity to those of Class I ribozymes. A
series of kinetic
analyses were used to compare the characteristics of the Class I and Class
VIII motifs. In each case,
the bimolecular constructs depicted in Figure 14 were used for comparison.
Each of these
substrates (trace) can be saturated with ribozyme as depicted in Figure 15a.
Furthermore, each
motif has near identical characteristics upon variation of monovalent salt
(Figure 15b), pH (Figure
15c), and divalent magnesium (Figure 15d). These results support the view
that, at their respective
core, the Class I and Class VIII motifs are similar.
Diagnostic uses
Enzymatic nucleic acids of this invention can be used as diagnostic tools to
examine genetic
drift and mutations within diseased cells or to detect the presence of target
RNA in a cell. The close
relationship between enzymatic nucleic acid activity and the structure of the
target RNA allows the
detection of mutations in any region of the molecule which alters the base-
pairing and three-
dimensional structure of the target RNA. By using multiple enzymatic nucleic
acids described in
this invention, one can map nucleotide changes which are important to RNA
structure and function
in vitro, as well as in cells and tissues. Cleavage of target RNAs with
enzymatic nucleic acids can
be used to inhibit gene expression and define the role (essentially) of
specified gene products in the
progression of disease. In this manner, other genetic targets can be defined
as important mediators
of the disease. These experiments can lead to better treatment of the disease
progression by
affording the possibility of combinational therapies (e.g., multiple enzymatic
nucleic acids targeted
to different genes, enzymatic nucleic acids coupled with known small molecule
inhibitors, or
intermittent treatment with combinations of enzymatic nucleic acids and/or
other chemical or
biological molecules). Other in vitro uses of enzymatic nucleic acids of this
invention are well
known in the art, and include detection of the presence of mRNAs associated
with disease
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
57
condition. Such RNA is detected by determining the presence of a cleavage
product after treatment
with a Enzymatic nucleic acid using standard methodology.
In a specific example, enzymatic nucleic acids which can cleave only wild-type
or mutant
forms of the target RNA are used for the assay. The first enzymatic nucleic
acid is used to identify
wild-type RNA present in the sample and the second enzymatic nucleic acid can
be used to identify
mutant RNA in the sample. As reaction controls, synthetic substrates of both
wild-type and mutant
RNA can be cleaved by both enzymatic nucleic acids to demonstrate the relative
enzymatic nucleic
acid efficiencies in the reactions and the absence of cleavage of the "non-
targeted" RNA species.
The cleavage products from the synthetic substrates will also serve to
generate size markers for the
analysis of wild-type and mutant RNAs in the sample population. Thus each
analysis can involve
two enzymatic nucleic acids, two substrates and one unknown sample which can
be combined into
six reactions. The presence of cleavage products can be determined using an
RNAse protection
assay so that full-length and cleavage fragments of each RNA can be analyzed
in one lane of a
polyacrylamide gel. It is not absolutely required to quantify the results to
gain insight into the
expression of mutant RNAs and putative risk of the desired phenotypic changes
in target cells. The
expression of mRNA whose protein product is implicated in the development of
the phenotype is
adequate to establish risk. If probes of comparable specific activity are used
for both transcripts,
then a qualitative comparison of RNA levels will be adequate and will decrease
the cost of the initial
diagnosis. Higher mutant form to wild-type ratios will be correlated with
higher risk whether RNA
levels are compared qualitatively or quantitatively.
Additional Uses
Potential uses of sequence-specific enzymatic nucleic acid molecules of the
instant invention
include many of the same applications for the study of RNA that DNA
restriction endonucleases
have for the study of DNA (Nathans et al., 1975 Ann. Rev. Biochem. 44:273).
For example, the
pattern of restriction fragments can be used to establish sequence
relationships between two related
RNAs, and large RNAs can be specifically cleaved to fragments of a size more
useful for study. The
ability to engineer sequence specificity of the enzymatic nucleic acid is
ideal for cleavage of RNAs
of unknown sequence.
The nucleic acid catalysts of the instant invention can be used to
specifically cleave an RNA
sequence for which an appropriately engineered nucleic acid catalyst base
pairs at the designated
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
58
flanking regions (e.g., X and Y in Formulae I - IV). Suitable target RNA
substrates include viral,
messenger, transfer, ribosomal, nuclear, organellar, other cellular RNA, or
any other natural RNA
having a cleavage sequence, as well as RNAs, which have been engineered to
contain an appropriate
cleavage sequence. The nucleic acid catalysts are useful in vivo in
prokaryotes or eukaryotes of
plant or animal origin for controlling viral infections or for regulating the
expression of specific
genes.
Once introduced into the cell, the nucleic acid catalyst binds to and cleaves
the target RNA
sequence or sequences for which it has been designed, inactivating the RNA. If
the RNA is
necessary for the life cycle of a virus, the virus will be eliminated and if
the RNA is the product of a
specific gene, the expression of that gene will thus be regulated. The nucleic
acid catalyst can be
designed to work in prokaryotes and within the nucleus (without poly(A) tail)
or in the cytoplasm of
a eukaryotic cell (with polyadenylation signals in place) for plants and
animals.
All patents and publications mentioned in the specification are indicative of
the levels of skill
of those skilled in the art to which the invention pertains. All references
cited in this disclosure are
incorporated by reference to the same extent as if each reference had been
incorporated by reference
in its entirety individually.
One skilled in the art would readily appreciate that the present invention is
well adapted to
carry out the objects and obtain the ends and advantages mentioned, as well as
those inherent
therein. The methods and compositions described herein as presently
representative of preferred
embodiments are exemplary and are not intended as limitations on the scope of
the invention.
Changes therein and other uses will occur to those skilled in the art, which
are encompassed within
the spirit of the invention, are defined by the scope of the claims.
It will be readily apparent to one skilled in the art that varying
substitutions and modifications
may be made to the invention disclosed herein without departing from the scope
and spirit of the
invention. Thus, such additional embodiments are within the scope of the
present invention and the
following claims.
The invention illustratively described herein suitably may be practiced in the
absence of any
element or elements, limitation or limitations which is not specifically
disclosed herein. Thus, for
example, in each instance herein any of the terms "comprising", "consisting
essentially of", and
"consisting of may be replaced with either of the other two terms. The terms
and expressions
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
59
which have been employed are used as terms of description and not of
limitation, and there is no
intention that in the use of such terms and expressions of excluding any
equivalents of the features
shown and described or portions thereof, but it is recognized that various
modifications are possible
within the scope of the invention claimed. Thus, it should be understood that
although the present
invention has been specifically disclosed by preferred embodiments, optional
features, modification
and variation of the concepts herein disclosed may be resorted to by those
skilled in the art, and that
such modifications and variations are considered to be within the scope of
this invention as defined
by the description and the appended claims.
In addition, where features or aspects of the invention are described in terms
of Markush
groups or other grouping of alternatives, those skilled in the art will
recognize that the invention is
also thereby described in terms of any individual member or subgroup of
members of the Markush
group or other group.
Thus, additional embodiments are within the scope of the invention and within
the claims that
follow.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
a~ d
H C C U U N E- O N U U N
. .
Q ~ ~ ~ ~ = N U
Q
O N O O lf~tf7 O
f~ f~~t7lON ~ M Z ~ ~ ~ ~ LO~ ~ M Z
~
N N
41 d
-
~ .
U U N N ~ , ~ ~ U U O O N
.,
d
N O tntnO ~ M M O N cnN O
~
Q Q
~ M Z ~ N N ~ ~ M Z
O O
y, N N In tnN ~ ~, ~ t(7
C C
Z ~ Z
L ~ L
_ d _ d
N ~
~ ~
U U
f- U U U U N ~ H U U U U O
O N U U O O U ~ N O U U N N U
M . n tn cncn ~"~= u mnO O tntn
O c N N O Q O N N O Q
U N
~ LO I ~ - Z m ~ In Intn lf7O ~ Z
' d' t
tf~n N ~ r a ~ ~ ~ ~
d d
() ~ J J J J J J J ~ ()~ J J J J J
Q1 O ~ s ~ ~ ~ ~ ~. N O i s ~ Z ~
N ~ ~ M M M M ~ ~ N N M
_
fl
' c' ~ d' ~ N ~tM
N Q '7 Q M M I'
~ N N N N ~ CO Cfl= ~ ~ N N N
w
C C
N
O ~ O r
~
G7 d
tf7 ~ N
N O ~ O (O(ON ~ O 3 ~ ~ ~ O
~ M O COf~r-N Q ~ M 00~ N O O ~ Q
Q LLI CO N ~ ~ ~-~ '~ Z m 111 e- M CO - f~N I~ Z
N N
O O ~ O O O
' N '~ ~ N
O - -O_
O
O ~ . ~ O
~
N .~ ~ O L N
O f"C ~, 07 ~_ ~' O ~ C ~, 07
C Q . C Q .
L L_ (0 C L L N C
V V
07 t~ ~ O C 7 ~ ~ ~ ~ N c j O
. .
~ U ~ ~ U ~ ~ V ~ ~ U
41 L U O d U N
Q Z H-o m Q ~ ~ c~Q Z H ~ m Q
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
61
0
.Q
a
d
U U
O N N O
O O N ~ N N O
a
M c~ ~ O ~ O N
M Z
N
d
f~ C U
_ N
' o o O O ~ ~ O a
a ~ncn~nN
~
ao 0o O O m O O
M N Z
d
C)
'= N i a a
Q
t c c
n n n O
CO f0 ~ r M ~ Z
O
J
T
J
O r
E J J J J O J
~ ~
' .
O O 3.Z ~ ~ N O
O
r ~ ~ ~ O O ~ r
C l l p
N p
3 O O O O ~ O N
~ c~o~ ~ o m ~ ~ d
n
EZ 0
QD ~ ~ 1~1~N 00 0 r
0 .d
C1
C
O
'O
d
s :r
r
. .r
0
N ~ N ~ l
~ 0
i' O COO I~ w'
C a0
~ N t'~ O
lI~N lI~~ Ln C7
N M N ~ ' ~ ~
O ~ G7
~ CD
111 N ~ N ~ N CO M Z v
D C
_
r
O O
C
O
' N
O a O
N ~ d
~ . E
~
d
fl-~ c~~ N V p ar
7
a o a C co~ ca
Q
z
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
62
I
ri~ M~d'~t.n~l0t~~00~01O r~N M ~'L!7l0L~0001O n-IN M
N~
W -1r-Ir-Ir1rir1rir1r1N N N N
4~
O
O
U > ~'
U O U
Q O Q
U U
C7H H H U C7
C7U U U H C7
H
H ~ ~ ~ U L7H C7L7C7C7U C7FCC7C7FC C7~ U
7 C7H H H H U H U H H H H H C7U H H H
C C7C7C7~ H U U U H H H H U H H H H U rCU U U
H
C7FCr~CFCC7H U H H H U U U H U U FCFCC7U H H C7
c/1 U H H H r.~C7H H H H H H H H H H H H H H H H C7
d U H H H H H C7C7C7U U U U L7U C7H C7U U C7C7H
C7C7L7z7~CH H H U U H U H H H C7H H H H
7 7 7 7 7 7 7 7 C U ~ ~ 7 7 H
H C7C7C7C7U C U C C C ~CC C C C ~ U H H C C r.~
H H H H H H H H H H H C7 H H
V C7U U H H H r.~~ rCr.~r.~C7r.~r.~r.~r.~U C7C7r.~r.~r.~C7
U H H H H H C7C7C7C7C7U C7C7C7C7H H U U C7C7C7
U U U U FCH H H H H r.~H H H H U H aCH H H H
C7H H H H C7C7C7C7L7C7C7C7L7C7L7H C7C7C7C7C7H
H C7C7C7C7H H H H H H H H H H H C7H H H H H r.~
H H H ~ H H H H H
.7.77 .7E''~ ~ ~ ~ H ~ ~ ~ ~ C7 ~ ~
L C C
L7L7C7C7C7C7 ~ C7 C7C7C7L7 H
H U U U H H H H H H H C7
FCC7C7C7C7FCC7C7C7C7C7FCC7L7C7C7C7FCFC~ C7C7H
C7L7C7C7C7C7C7C7C7C7C7C7C7C7C7L7C7C7C7L7C7C7C7
r.~H H H H FCC7H C7C7C7r~C7C7H L7H FCFCFCH H H
H H H H H H U U U U U H U U U U H H H H U U H
H C7C7C7C7H H H H H H H H H H H C7H H H H H U
G7 U H H H H U U U U U U U U U U U H U U U U U H
> ~CU U U U ~CFC~C~C~C~C~C~C~C~C~CU FC~C~CFCFC~C
H H H H H H H H H H H H H H H H H H H H H H H
v H H H H H H H H H H H H H H H H H H H H H H H
H H H H H H H H H H H H H H H H H H H H H H H
C7C7C7C7C7C7C7C7C7C7C7C7C7C7C7C~C~C~C7C7C7C7C7
U U U U U U U U U U U U U U U U U U U U U U U
C7C7C7ChChC7C7C7L7C7C7C7C7C7C7C7C7C7C7C7C7L7L7
V ~ FCFCFCFCFC~CFCFCFCFC~ FCFC~C~CFCFC~C~CFCFCFC
C C7C7C7C7c7L7C7C7L7C7C7C7C7C7C7C7C7C7C7C7C7C7L7
C7C7C7C7C7L7C7C7C7C7C7C7C7C7C7C7C7C7C7C7C7L7L7
7 C7FCFC~ FCC7C7C7C7C7C7C7C7C7C7C7rCC7C7C7C7C7~C
C7C7C7C7t7C7C7C7t7C7C7L7C7C7C7C7C7C7C7C7C7L7L7
G~ H H H H H H H H H H H H H H H H H H H H H H H
(n U U U U U U U U U U U U U U U U U U U U U U .U
C7ChC7C7C7C7C7U C7C7C7C7C7C7C7C7L7L7C7C7L7C7Ch
N ~C~CFC~C~C~C~C~C~C~C~C~C~C~C~C~C~C~ FC~C~C~C~C
U U U U U U U U U U U U U U U U U U U U U U U
C7C7C7C7C7C7C7C7C7C7C7C7C7C7U C7C7L7C7C7C7C7C7
H H H H H H H H H H H H H H H H H H H H H H H
H- C7C7C7C7C7C~C7C7C7C7C7C7C7C7C7C7L7L7C7C7U L7C7
M
00
M V1~ Q~O O 00d'~ 00.~l~00O 00
~
N N N N ~ N M ~ I~~ ~ ~ ~nV~V~~O~ ~Ot
O ~ ~ ~ ~ ~ ~ ~ ~ N ~ ~ i ~ ~
-, ~ ~ ~ ~ ~ .-r.-. ....-,.~, ,-..-..-..-,.-~
r,, N N N i N N N ~ N N N N N N N i N N
~ ~ 7 7 7 7 7 ~ ~ 7 7 ~ 7 7 7 7 7
U U C7C7C7C C N C C C U C C C C C C N C C
~
N
a~
v
o" ~~
U
U
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
63
d'L(1l0L OD01O riN M d'L(1l0f~CO01O v-IN M '1
N N N N N N M M M M M M M M M M ~ d'd~d'V
N G
O
O -rl
E ~',
-ri JJ
~
C_ C_~ C_
E E ~, ~ C
v
N 4) M ~ 0~
> > N
p V V p p p p G
O Q Q O O O O ~ o
H H H H H H rC o
i
C7C7H U C7L7L7C7C7C7C7U ~C~
C7C7U H C7U U U U U U U U ~y
U U U U U U U U U ~
C7 H H H H C7H C
7
H C7H U C7C7H H H ~ ~ ~ ~ ~ ~ H C7~ v
H H U U C7C7H C7 FCH L7U H '~
H U H H H H C7~ C7C7C7C7L7C7C7H U H U r.~~
H H FCH C7C~C7H H C7C7C7C7C7C7L7C7H L7H v
H H H H H U U U U H U U H
C7L7 C7FCU U H C
7
~ H H L7C7C7L7C7U H H H H H H C7H C7H C7~,
H ~ H U U H ~ U U N H U H H ~ U
C7L7C C C7b
7 7
L7~CH C7H H FCU U H H H H H H C7L7C7C7C7~
H C7~CH U U H ~CU H H U U H H C7C7C7C7H
U
L7H H C7H H U U H L7C7C7C7C7C7C7C7C7C7H
H L7C7H H H C7C7H L7C7C7C7L7C7H H H H U ~,
H H H H r.G C7H H H H H H H H H H C7
H ~C~CH ~ FCL7L7C7C7L7C7U U U U H
~ ~
H H C7C7C7H H H H H H H H H H H
~
C7C7H C7C7L7L7C7C7C7C7U U U U C7
LhC7 C7C7C7H C7C7H H H H H H C7L7C7C7C7-~
~
L7C7 C7C7C~C7C7~CC7C7C7C7L7C7C7L7C7C7Chrn ~
LhL~H H FCH C7H C7L7C7C7~ v U
H H C7H r.~H r.~U ~ ~ ~ ~ ~ ~ ~ U U U
~
U U H U U U U r.~C7H H H H H U C7~ ~ ~ H
~CFCU ~CC7C7FCC7C7FC~C~C~C~C~C~C ~Co
H H H H U U C7H H H H H H C7C7L7C7U v
~ ~
H H H H C7C7L7rCFCFCFCFCFCC7C7C7C7H
H H H H C7L7 H L7H U U U U H U U U U H
C7C7C7C7L7C~~ FCL7C7C~C7L7C7~7L7C7C7C7r.~~
U U U U L7Z7C7H L7H H U H U H r.~FCFCr.~U ~
C7C7C7C7C7L7C7H rCU U U H U U C7U C7C7C7~
Ch~ L7C7H H C7U H r.~r.~~Cr.~FC~CC7L7C7L7U
~CC7C7C7H U C7L7C7C7C7C7H H H H C7''' v
'
C7L7C7C7C7L7H ~ ~ U U U U U U H H H H C7v o ~
C7L7C7L7C7C7L7U H rCrCr.~r~rCFCH H H H U
C7L7~CL"J~C~CFCC7H C7C7C7C7C7C7~ rC~C~CE-'~
L7C7C7L7C7C7H H C7C7U C7C7C7C7L7C7C7C7,
v v ~
H H H H H H H ~ H r.~r.~r.~,~C~ r.~C7L7C7C7H
U U U U U U C7C7C7U U U U U U ~ r.~r.~FCC7.
~~
C7c7C7C7U U U C7C7C7C7L7C7C7L7H H H H L7,
a o 0
~C~ ~C~ ~ ~ c7~C~ ~C~C~C~CC7C7C7C7H U
7 7 7 7 7
U U U U C7C7~CC7C7H H H H H H C C C C L ,~
C7C7L7C7 C7 C7~ ~ ~ ~ ~ ~ C7C7C7C7L7
E-iH H H C7C7C7L7L7 C7C7C7C7FCr, 3
,--i
~ ~ ~ ~ U U U ~ ~ U
7 _7_7_7C L C L7H H H H H H C77 C7FC~ov
C C L C 7 7 7 L
u~ rt
E
N ~ M ~n
~ ~ ~ ~
N ~ N o0 N I~ .~ O O~t~~nooN ~n~
t~o00000 ~ ~ N ~n ~Ol~00~ v~~nl~N ~
i i i i M i ~ ~ ~ M i ~ i i i i i i i i r-I O
U7 t
.-,.~.--n.-, .-r~ ~ ,-m --i~ ,~~ ~ ~ ~ ~ f.~ U
4: M
N N N N ~ N N . N ~ N ~ N N N ~ N N N ~ N C
U'CTU''UN 'UC7N C7N C7N CJCJCJC7C7CJC7'U.b O
~ to
C7 CJ CJ CJ
N ~ 'CS
t~
?-i O
G
~a o
x
fJ) U
p,
W 1J
i"'i ~ ~ ~ ~ U ~ O
W
Sa -r-1
LL
N 1J
J-1
W
U1 U
x
U U U U U ~ v ~
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
64
Table 3: Kinetic unimolecular parameters of selected clones
Observed Amount
CIaSS Clone Cleavage site rate constant Processed
I 23 nt 4 nd Nd
27 nt 4 0.024 80%
51 nt 4 0.0048 nd~
II 37 nt 6 0.0074 70%
47 nt 6 nd Nd
53 nt 6 nd Nd
III 21 nt 4 0.014 7%
IV 32 nt 7 0.093 50%
V 52 nt 1 0.014 70%
VI 33 Inactive nd nd
34 nt 7~ nd nd
Reaction conditions: 0.5 mM MgCl2, 7 mM putrescine, 10 mM glutathione, SO
mM HEPES pH 7.6, 37°C.
Cleavage site: enzyme cleaves after the indicated nucleotide, numbered 1 to 13
(Figure 1) within the RNA region.
Observed rate constant is in units of mine.
Amount processed is the amount of cleavage at which the reaction plateaus.
*Reaction kinetics changed during the time course such the new reaction did
not
asymptotically approach a minimum.
~Enzyme had reacted prior to assay and did not react further. Presumably it
reacted during elution from the purification gel but these conditions could
not be
duplicated.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
Table 4: Relative cleavage rates of Class I DNAzyme motif on
various substrates
Substrate Substrate sequence Relative SEQ ID NO
name rate of
cleavage*
Model, FigureaagagUAA-UAA-UGAAAGCag1 44
2
Figure Sa, GAGGGUAU-UAA-UAAUGAAAG0.18 45
site 1
Figure Sa, GGUAUUAA-UAA-UGAAAGCUA0.30 46
site 2
Figure Sb, GGGAAUGC-UAA-UCUACUACG0.0057 47
site 1
Figure Sb, CUACUACG-UAA-UCAGCAACG0 48
site 2
Figure 5b, CAGCAACG-UAA-UGCUCUGUA0.011 49
site 3
Figure Sb, GCUCUGUA-UAA-UGACGACUG0 I 50
site 4 I I
Legend: The catalytic core from G1 to T19 shown in Figure 2 was
synthesized between arms that base-pair to the substrate sequence such
that the U immediately 3' to the cleavage site is engaged in a wobble pair
with G1 of the catalytic core and the UAA separately by hyphens is
bulged. Cleavage site is 3' to the U of the bulged UAA. Substrates are
RNA except for the model substrate where DNA is indicated by
lowercase.
Assay conditions: 50 ~tM enzyme, trace (<5) nM substrate, 2 mM Mg2+,
150 mM K+, 7 mM putrescine, pH 7.6, 37°C
* This data is meant for comparison only. Assays for the substrate sites in
Figure
5B are not multiple-point rate assays and the rate constant is estimated from
a
single time point. For this reason, the actual rate constants or estimated
rate
constants have been normalized to the rate for the model substrate so that the
actual values will not be emphasized.
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
66
Table 5: Class V ribozyme 2'-O- methyl arm optimization
RPI No. DESCRIPTION ~ obs min. I) % in 1s'
exponential
14880 6 7 2'-O-Me arms0.066 82.3
14881 5 ,7 2'-O-Me 0.066 81.0
arms
14189 WT all ribose 0.044 72.5
"bulged U"
14882 S G 2'-O-Me arms0.043 76.6
14883 5 5 2'O-Me arms 0.037 75.4
14885 4 4 2'-O-Me arms0.031 84.0
14884 4 5 2'-O-Me arms0.030 82.8
14879 6 8 2'-O-Me arms0.0009 60.0
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
67
Table 6: Class V ribozyme motif 2'-O-methyl core
"wal k"
RPI No. DESCRIPTION k obs ~min.-~)Krel to .20672
14189 WT All Ribose 0.036 0.64
Assa Control
14880 6 7 2'-OMe Arms 0.061 1.09
e1- urified
15297 6 7 2'-OMe Arms 0.056 / 0.058 1.00
crude s nthesis
15305 G 7 2'-OMe Arms 0.072 1.29
+
Core 2'-OMe A6
15310 7 7 2'-OMe Arms 0.070 1.25
ribo core
15309 6 7 2'-OMe Arms 0.069 1.23
+
Core 2'-OMe G2
15299 6 7 2'-OMe Arms 0.068 1.21
+
Core 2'-OMe A12
15300 G c 7 2'-OMe Arms0.060 1.07
+
Core 2'-OMe All
15306 6 7 2'-OMe Arms 0.057 1.02
+
Core 2'-OMe A5
15301 6 7 2'-OMe Arms 0.050 0.89
+
Core 2'-OMe G10
15298 6 7 2'-OMe Arms 0.030 0.54
+
Core 2'-OMe G13
15303 6 7 2'-OMe Arms 0.029 0.52
+
Core 2'-OMe G8
15304 6 7 2'-OMe Arms 0.017 0.30
+
Core 2'-OMe C7
- 15308 6 7 2'-OMe Arms 0.010 0.17
+
Core 2'-OMe A3
15302 6 7 2'-OMe Arms 0.0004 0.007
+
Core 2'-OMe U9
15307 G 7 2'-OMe Arms 0.0001 0.002
+
Core 2'-OMe U4
15311 All 2'-OMe 0.00008 0.001
A14.1 = Ribo
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
68
Table 7: Class V ribozyme motif combination core
RPI No. DESCRIPTION k o6, (min:') k,e, to .20672
(all other residues All Ribo Core
= 2'-
O-Meth 1
15705 7 RIBO CORE 0.127 2.31
A3, U4, C7, G8,
U9, G13,
A14.1 = ribo
15297 ALL RIBO "CORE" 0.055 1.00
G2 - A14.1 = ribo
15702 6 RIBO CORE 0.036 0.65
U4, C7, G8, U9,
G13, A14.1
= ribo
15700 4 RIBO "CORE" 0.0019 0.03
U4, C7, U9, A14.1
= ribo
15701 5 RIBO "CORE" 0.0012 0.02
U4, C7, G8, U9,
A14.1
= ribo
15703 3 RIBO CORE 0.0006 0.01
U4, U9, A14.1 =
ribo
15704 3 RIBO CORE 0.0006 0.01
U4, U9, A14.1 =
ribo
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
69
Table 8: Class V ribozyme motif with pyrimidine stabilized core
RPI NO. DESCRIPTION k abs (min.-')k~~i to .21422k~ei to
.20b
(7 Ribo Core)(All Ribo
C
15705 7 RIBO CORE 0.113 1.00 2.05
A3, U4, C7, G8,
U9, G13,
A14.1 = ribo
16188 "7 ribo" 0.144 1.28 2.62
U4 = PS2
16191 "6 ribo" 0.121 1.07 2.2
C7 = 2'F-C
16197 "6 ribo" 0.063 0.56 1.15
C7 = 2-NH2-C
16186 "7 ribo" 0.063 0.56 1.15
U9 = PS2
16194 "6 ribo" 0.030 0.27 0.55
C7 = 2-deoxy-C
16196 "6 ribo" 0.025 0.22 0.45
U9 = 2-NH2-U
16192 "6 ribo" 0.015 0.13 0.27
U9 = 2'F-U
16195 "6 ribo" 0.011 0.10 0.20
U9 = 2-deoxy-U
16198 "6 ribo" 0.001 0.009 0.02
U4 = 2-NH2-U
16190 "6 ribo" 0.0005 0.004 0.009
U4 = 2'F-U
16193 "6 ribo" 0.0002 0.002 0.004
U4 = 2'-deoxy-U
PS2 = phosphorodithioate linkage
2'-F-U = 2'-deoxy-2'-fluoro uridine
2'-F-C = 2'-deoxy-2'-fluoro cytidine
2'-NH2-U = 2'-deoxy-2'-amino uridine
2'-NH2-C = 2'-deoxy-2'-amino cytidine
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
Table 9: Class V ribozyme motif with reduced ribo content
RPI NO DESCRIPTION k obs kr~~ to .21422kre~ to .20672
(min.-') (7 Ribo Core)(All Ribo
Core;
15705 7 RIBO CORE
A3, U4, C7, G8, 0.117 1.00 2.13
U9, G13,
A14.1 = ribo
16013 4 RIBO CORE
U4, G8, U9, A14.1 0.041 0.35 0.75
=
ribo
17584 4 RIBO
U4, U9 = PS 0.0008 0.007 0.02
C7 = 2'-OMe
17585 5 RIBO
U4, U9 = PS 0.0018 0.02 0.03
C7 = 2'-OMe
17586 4 RIBO
U4 = PS, U9 = NH2 0.0003 0.003 0.005
C7 = 2'-OMe
17587 5 RIBO
U4 = PS, U9 = NH2 0.0006 0.005 0.01
C7 = 2'-OMe
17589 5 RIBO
U4 = PS, U9 = NH2 0.0021 0.02 0.04
C7 = 2'-F
17590 7 RIBO
C7 = PS 0.105 0.90 1.91
17591 6 RIBO
U4, U9 = PS 0.022 0.19 0.40
C7 = 2'-OMe
17588 4 RIBO
U4, U9 = PS 0.012 0.10 0.22
C7=2'F
PS = phosphorothioate linkage
2'-F = 2'-deoxy-2'-fluoro
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
71
Table 10: Class V ribozymes
Synth . SEQ RPI Ribozyme
No. ID No.
#
19984 51 14880 gga guaA GA CGUGAAG Au caggacB
UAA
19985 52 14881 gga guAA GA CGUGAAG Au caggacB
UAA
19120 53 14189 GGA GUA AGA CGUGAA AU CAGGAC
UAA G
19986 54 14882 gga guAA GA CGUGAA AU caggacB
UAA G
19987 55 14883 gga guAA GA CGUGAAG AU CaggacB
UAA
19989 56 14885 gga gUAA GA CGU AU CAggacB
UAA GAA
G
19988 57 14884 gga gUAA GA CGUGAAG AU CaggacB
UAA
19983 58 14879 gga guaA GA CGUGAAG au caggacB
UAA
20672 59 15297 gga guaA GA CGUGAAG Au caggacB
UAA
20680 60 15305 gga guaA GA CGUGAAG Au caggacB
UAa
20685 61 15310 gga guaa GA CGUGAAG Au caggacB
UAA
20684 62 15309 gga guaA GA CGUGAAG Au caggacB
UAA
20674 63 15299 gga guaA GA CGUGAaG Au caggacB
UAA
20675 64 15300 gga guaA GA CGU Au caggacB
UAA GaA
G
20681 65 15306 gga guaA GA CGUgAA Au caggacB
UaA G
20676 66 15301 gga guaA GA CGUGAAG Au caggacB
UAA
20673 67 15298 gga guaA GA CGUGAAg Au caggacB
UAA
20678 68 15303 gga guaA GA CgUGAAG Au caggacB
UAA
20679 69 15304 gga guaA GA cGUGAA Au caggacB
UAA G
21076 70 15308 gga guaA GA CGUGAAG Au caggacB
UAA
20677 71 15302 gga guaA GA CGuGAAG Au caggacB
UAA
21075 72 15307 gga guaA GA CGUGAAG Au caggacB
uAA
20686 73 15311 gga guaa ga cgugaag Au caggacB
uaa
21422 74 15705 gga guaa gA CGUgaaG Au caggacB
Uaa
21577 75 15702 gga guaa ga CGUgaaG Au caggacB
Uaa
21417 76 15700 gga guaa ga CgUgaag Au caggacB
Uaa
21418 77 15701 gga guaa ga CGUgaag Au caggacB
Uaa
21420 78 15703 gga guaa ga cgUgaag Au caggacB
Uaa
21421 79 15704 gga guaa ga cgUgaag Au caggacB
Uaa
22128 80 16188 gga guaa CGU cag
gA US2aa gaa gacB
G
Au
22131 81 16191 gga guaa gA CGUgaaG Au caggacB
Uaa
22137 82 16197 gga guaa gA CGUgaaG caggacB
Uaa Au
22126 83 16186 gga guaa gaa cag
gA Uaa G gacB
CGUS2 Au
22134 84 16194 gga guaa gA CGUgaaG Au caggacB
Uaa
22136 85 16196 gga guaa gA CGUgaaG Au caggacB
Uaa
22132 86 16192 gga guaa gA CGUgaaG Au caggacB
Uaa
22135 87 16195 gga guaa gA CGUgaaG Au caggacB
Uaa
22138 88 16198 gga guaa gA CGUgaaG Au caggacB
Vaa
22130 89 16190 gga guaa gA CGUgaaG Au caggacB
Uaa
22133 90 16193 gga guaa gA CGUgaaG Au caggacB
Uaa
21818 91 16013 gga guaa ga cGUgaag Au caggacB
Uaa
23786 92 17584 gga guaa cGUSgaa caggacB
ga Usaa g
Au
23787 93 17585 gga guaa cGUSgaa caggacB
ga Usaa G
Au
CA 02398750 2002-07-30
WO 01/59102 PCT/USO1/04223
72
23788 94 17586 gga guaaga UsaacGU gaag AucaggacB
23789 95 17587 gga guaaga UsaacGU gaaG AucaggacB
23791 96 17589 gga guaaga UsaaCGU gaaG AucaggacB
23792 97 17590 gga guaaga Uaa gaaG caggacB
CSGU Au
23793 98 17591 gga guaaga UsaacGUSgaaG AucaggacB
24021 99 17588 gga guaaga UgaaCGUggaag AucaggacB
I
U, C = 2'-deoxy-2'-fluoro Uridine, Cytidine
U, C = 2'-deoxy Uridine, Cytidine
U, C = 2'-deoxy-2'-amino Uridine, Cytidine
UPPER CASE = RIBO
lower case = 2'-O-Methyl
S = phosphorothioate linkage
S2 - phosphorodithioate linkage
B = 3',3'-inverted deoxyabasic moiety
