Note: Descriptions are shown in the official language in which they were submitted.
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HAMMERHEAD RIBOZYMES WITH EXTENDED CLEAVAGE RULE
Background of the Invention
The present invention is in the field of compositions having RNA-
cleavage activity.
Hammerhead ribozymes are an example of catalytic RNA molecules
which are able to recognize and cleave a given specific RNA substrate
(Hotchins et al., Nucleic Acids Res. 14:3627 (1986); Keese and Symons, in
Viroids and viroid - like pathogens (J.J. Semanchik, publ., CRC-Press, Boca
Raton, Florida, 1987), pages 1-47). The catalytic center of hammerhead
ribozymes is flanked by three stems and can be formed by adjacent sequence
regions of the RNA or also by regions which are separated from one another
by many nucleotides. Figure 1 shows a diagram of such a catalytically active
hammerhead structure. The stems have been denoted I, II and III. The
nucleotides are numbered according to the standard nomenclature for
hammerhead ribozymes (Hertel et al., Nucleic Acids Res. 20:3252 (1992)).
In this nomenclature, bases are denoted by a number which relates their
position relative to the 5' side of the cleavage site. Furthermore, each base
that is involved in a stem or loop region has an additional designation (which
is denoted by a decimal point and then another number) that defines the
position of that base within the stem or loop. A designation of N"~' would
indicate that this base is involved in a paired region and that it is the
third
base in that stem going away from the core region. This accepted
convention for describing hammerhead derived ribozymes allows for the
nucleotides involved in the core of the enzyme to always have the same
number relative to all of the other nucleotides. The size of the stems
involved in substrate binding or core formation can be any size and of any
sequence, and the position of A~, for example, will remain the same relative
to all of the other core nucleotides. Nucleotides designated, for example,
' N"'2 or Ny" represent an inserted nucleotide where the position of the caret
(") relative to the number denotes whether the insertion is before or after
the
indicated nucleotide. Thus, N"'2 represents a nucleotide inserted before
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nucleotide position 12, and N9" represents a nucleotide inserted after
nucleotide position 9.
The consensus sequence of the catalytic core structure is described by
Ruffner and Uhlenbeck (Nucleic Acids Res. 18:6025-6029 (1990)). Perriman
et al. (Gene 113:157-163 (1992)) have meanwhile shown that this structure
can also contain variations, for example, naturally occurring nucleotide
insertions such as N9" and N"'z. Thus, the positive strand of the satellite
RNA of the tobacco ring-spot virus does not contain any of the two
nucleotide insertions while the +RNA strand of the virusoid of the lucerne
transient streak virus (vLTSV) contains a N9" = U insertion which can be
mutated to C or G without loss of activity (Sheldon and Symons, Nucleic
Acids Res. 17:5679-5685 (1989)). Furthermore, in this special case, N' = A
and R'S~' = A. On the other hand, the minus strand of the carnation stunt
associated viroid (- CarSV) is quite unusual since it contains both nucleotide
insertions, that is N"'z = A and N9" = C (Hernandez et al. , Nucleic Acids
Res. 20:6323-6329 (1992)). In this viroid N' = A and R'S~' = A. In
addition, this special hammerhead structure exhibits a very effective self
catalytic cleavage despite the more open central stem.
Possible uses of hammerhead ribozymes include, for example,
generation of RNA restriction enzymes and the specific inactivation of the
expression of genes in, for example, animal, human or plant cells and
prokaryotes, yeasts and plasmodia. A particular biomedical interest is based
on the fact that many diseases, including many forms of tumors, are related
to the overexpression of specific genes. Inactivating such genes by cleaving
the associated mRNA represents a possible way to control and eventually
treat such diseases. Moreover there is a great need to develop antiviral,
antibacterial, and antifungal pharmaceutical agents. Ribozymes have
potential as such anti-infective agents since RNA molecules vital to the
survival of the organism can be selectively destroyed.
One of the greatest impediments to using hammerhead based
ribozymes for pharmaceutical agents is the limited availability of acceptable
targets in normal Gerlach designs and when targeting pre-formed half
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hammerhead structures (see WO 97/18312). In addition to needing the
correct hybridizing sequences for substrate binding, substrates for
hammerhead ribozymes have been shown to strongly prefer the triplet
N'6.zU'6.'H" where N can be any nucleotide, U is uridine, and H is either
adenosine, cytidine, or uridine (Koizumi et al. , FEBS Lett. 228, 228-230
(1988); Ruffner et al., Biochemistry 29, 10695-10702 (1990); Perriman et
al., Gene 113, 157-163 (1992)). The fact that changes to this general rule
for substrate specificity result in non-functional substrates implies that
there
are "non core compatible" structures which are formed when substrates are
provided which deviate from the stated requirements. Evidence along these
lines was recently reported by Uhlenbeck and co-workers (Biochemistry
36:1108-1114 (1997)) when they demonstrated that the substitution of a G at
position 17 caused a functionally catastrophic base pair between G" and C3 to
form, both preventing the correct orientation of the scissile bond for
cleavage
and the needed tertiary interactions of C3 (Murray et al. , Biochem. J.
311:487-494 (1995)). The strong preference for a U at position 16.1 may
exist for similar reasons. Many experiments have been done in an attempt to
isolate ribozymes which are able to efficiently relieve the requirement of a U
at position 16.1, however, attempts to find hammerhead type ribozymes
which can cleave substrates having a base other than a U at position 16.1
have proven impossible (Perriman et al., Gene 113, 157-163 (1992)).
Efficient catalytic molecules with reduced or altered requirements in
the cleavage region are highly desirable because their isolation would greatly
increase the number of available target sequences that molecules of this type
could cleave. For example, it would be desirable to have a ribozyme variant
that could efficiently cleave substrates containing triplets other than
N'6.zU'6.'Ii" since this would increase the number of potential target
cleavage
sites.
Chemically modified oligonucieotides which contain a block of
deoxyribonucleotides in the middle region of the molecule have potential as
pharmaceutical agents for the specific inactivation of the expression of genes
(Giles et al., Nucleic Acids Res. 20:763-770 (1992)). These oligonucleotides
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can form a hybrid DNA-RNA duplex in which the DNA bound RNA strand
is degraded by RNase H. Such oligonucleotides are considered to promote
cleavage of the RNA and so cannot be characterized as having an RNA-
cleaving activity nor as cleaving an RNA molecule (the RNase H is
cleaving). A significant disadvantage of these oligonucleotides for in vivo
applications is their low specificity, since hybrid formation, and thus
cleavage, can also take place at undesired positions on the RNA molecules.
Previous attempts to recombinantly express catalytically active RNA
molecules in the cell by transfecting the cell with an appropriate gene have
not proven to be very effective since a very high expression was necessary to
inactivate specific RNA substrates. In addition the vector systems which are
available now cannot generally be applied. Furthelnore, unmodified
ribozymes cannot be administered directly due to the sensitivity of RNA to
degradation by RNases and their interactions with proteins. Thus, chemically
modified active substances have to be used in order to administer
hammerhead ribozymes exogenously (discussed, for example, by Heidenreich
et al., J. Biol. Chem. 269:2131-2138 (1994); Kiehntopf et al., EMBO J.
13:4645-4652 (1994); Paolella et al., EMBO J. 11:1913-1919 (1992); and
Usman et al., Nucleic Acids Symp. Ser. 31:163-164 (1994)).
U.S. Pat. No. 5,334,711 describes such chemically modified active
substances based on synthetic catalytic oligonucleotide structures with a
length of 35 to 40 nucleotides which are suitable for cleaving a nucleic acid
target sequence and contain modified nucleotides that contain an optionally
substituted alkyl, alkenyl or alkynyl group with 1 - 10 carbon atoms at the
2'-O atom of the ribose. These oligonucleotides contain modified nucleotide
building blocks and form a structure resembling a hammerhead structure.
These oligonucleotides are able to cleave specific RNA substrates.
The use of a large number of deoxyribonucleotides in the
hybridization arms or in the active center can lead to a loss of specificity
due
to an activation of RNase H since sequences which are related to the desired
target sequence can also be cleaved. Moreover, catalytic DNA oligomers are
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not particularly well suited for in vivo applications due to interactions with
.
proteins, and lack of resistance to degradation by nucleases.
It is therefore an object of the present invention to provide
compositions that cleave RNA, and in particular to provide RNA-cleaving
oligomers which at the same time have a high stability, activity, and
specificity.
It is another object of the present invention to provide compositions
that cleave RNA substrates having a cleavage site triplet other than
N'6.zUi6.iHn.
Summary of the Invention
Disclosed are compositions having an RNA-cleavage activity, as well
as their use for cleaving RNA substrates in vitro and in vivo. The
compositions contain an active center, the subunits of which are selected
from nucleotides and/or nucleotide analogues, as well as flanking regions
contributing to the formation of a specific hybridization with an RNA
substrate. Preferred compositions form, in combination with an RNA
substrate, a structure resembling a hammerhead structure. The active center
of the disclosed compositions is characterized by the presence of I'S~' which
allows cleavage of RNA substrates having C'6.'.
Brief Description of the Drawings
Figure 1 is a diagram of a hammerhead structure and the
corresponding nomenclature (SEQ ID NO:1). Cleavage occurs between H"
and N '.' to generate the 2' , 3'-cyclic phosphate at H".
Figure 2 is a diagram of an RNA substrate (SEQ ID N0:3) in
association with an example of an oligomer (SEQ ID N0:2) that cleaves the
RNA substrate. The structure formed by the oligomer and the substrate
resembles the structure of a hammerhead ribozyme. In this case, the
substrate makes up half of stems I and III, and loops I and III are not
present. Cleavage occurs 3' of H".
Figure 3 is a diagram showing the interaction of the A'S.'-U'6.' base
pair in hammerhead ribozymes {top), and the predicted isostructural
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interaction of a I'S~'-C'6.' base pair (bottom) that replaces the A'S.'-U'6.'
base.
pair.
Figure 4A is a graph of fraction of cleavage product versus time (in
minutes) for the cleavage of a short 5'-fluorescein labelled
oIigoribonucleotide substrate (SEQ ID N0:8) containing a GCA site by four
variants of 2'-O-allylated 5-ribo catalytic oIigomers each containing a
different nucleobase at position N' (U, C, A and G; SEQ ID NOS:18, 22,
23, and 24, respectively).
Figure 4B is a graph of fraction of cleavage product versus time (in
minutes) for the cleavage of a short 5'-fluorescein labelled
oligoribonucleotide substrate of SEQ ID N0:8 containing a GCA site by four
variants of 2'-O-allylated 5-ribo catalytic oligomers each containing a
different nucleobase or base analogue at position N' (U, 5-nitroindole, I, and
quinazoline-2, 4-dione; SEQ ID NOS:18, 27, 25, and 26, respectively).
Detailed Description of the Invention
Disclosed are compositions having an RNA-cleavage activity, as well
as their use for cleaving RNA-substrates in vitro and in vivo. The
compositions contain an active center, the subunits of which are selected
from nucleotides and/or nucleotide analogues, as well as flanking regions
contributing to the formation of a specific hybridization with an RNA
substrate. Preferred compositions form, in combination with an RNA
substrate, a structure resembling a hammerhead structure. The active center
of the disclosed compositions is characterized by the presence of I'S.' which
allows cleavage of RNA substrates having C'6.'.
All naturally occurring hammerhead ribozymes have an A's.'-U'6.~
base pair. In addition, it is known that substrates for ribozymes based on
the consensus hammerhead sequence strongly prefer a substrate that contains
an N'6.zU'6.'H" triplet in which H" is not a guanosine (Koizumi et al. , FEBS
Lett. 228, 228-230 (1988); Ruffner et al., Biochemistry 29, 10695-10702
(1990); Perriman et al., Gene 113, 157-163 (1992)). Many experiments have
been done in an attempt to isolate ribozymes which are able to efficiently
relieve the requirement of a U at position 16.1, however, attempts to find
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ribozymes which can cleave substrates having a base other than a U at
position 16.1 have proven impossible (Perriman et al., Gene 113, 157-163
1992, Singh et al., Antisense and Nucleic Acid Drug Development 6:165-168
( 1996)).
However, examination of the recently published X-ray crystal
structures (Pley et al., Nature 372:68-74 (1994), Scott et al., Cell 81:991-
1002 ( 1995), and Scott et al. , Science 274:2065-2069 ( 1996)) led to the
realization that the A's.'-U'6.' interaction is a non-standard base pair with
a
single hydrogen bond between the exocyclic amine (N6) of the adenosine and
the 4-oxo group of the uridine. Modeling studies (based on the crystal
structure) then ied to the discovery that the interaction of the wild-type
A'S.'-
U'6.' base pair can be spatially mimicked by replacement with an I'S''-C'b.l
base pair that adopts an isostructural orientation and which preserves the
required contact of the 2-keto group of C'~~' with A6 of the uridine turn. In
the model, the polarity of the stabilizing hydrogen bond between positions
15.1 and 16.1 is reversed in the I'S.'-C'6.' interaction, but the correct
orientation of the bases around this bond is maintained.
It has been discovered that Gerlach type ribozyme analogues
containing an inosine at position 15.1 readily cleave RNA substrates
containing an N16.2C16.1H17 triplet. Based on this, disclosed are
compositions,
preferably synthetic oligomers, which cleave a nucleic acid target sequence
containing the triplet N'6~ZC'6~'H". It is preferred that H" is not guanosine.
The ability to cleave substrates having N'6.zC'6.'Xn triplets effectively
doubles
the number of targets available for cleavage by compositions of the type
disclosed.
Compositions Having an RNA-cleavage Activity
Specifically disclosed is a composition that cleaves an RNA substrate,
where the composition includes components (a) and (b), where component (a)
includes a structure 5'-Z,-ZZ-3' and component (b) includes a structure
5'-Z3-Z4 3' . Components (a) and (b) can either be separate molecules or can
be covalently coupled. Elements Z, and Z4 in components (a) and (b) are
each oligomeric sequences which are made up of nucleotides, nucleotide
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analogues, or a combination of both, or are oligonucleotide analogues. The
oligomeric sequences of elements Z, and Z4 specifically interact with the
RNA substrate, preferably by hybridization.
In these preferred compositions, element ZZ has a structure ~ of
S'-X3X4XSX6X'X8X9-3', Or
s_X3X4XSX6X7X8X9X9"_3, ,
and element Z3 has a structure of
5~-X'ZX13X14x15.1-3~ or
,
5 ~-X~'ZX 12x 13X14X15.1-3' .
Elements Zz and Z3 in these preferred compositions are made up of
nucleotides, nucleotide analogues, or a combination of both. The nucleotides
and nucleotide analogues in elements ZZ and Z3 each have the structure
D
IS
(I)
In structure (I) each B can be adenin-9-yl, cytosin-1-yl, guanin-9-yl,
uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl, thymin-1-yl, 5-methylcytosin-1-yl,
2,6-diaminopurin-9-yl, purin-9-yl, 7-deazaadenin-9-yl, 7-deazaguanin-9-yl, 5-
propynylcytosin-1-yl, 5-propynyluracil-1-yl, isoguanin-9-yl, 2-aminopurin-9-
yl, 6-methyluracil-1-yl, 4-thiouracil-I-yl, 2-pyrimidone-1-yl, quinazoline-2,4-
dione-I-yl, xanthin-9-yl, NZ-dimethylguanin-9-yl or a functional equivalent
thereof;
Each V can be an O, S, NH, or CH, group.
Each W can be -H, -OH, -COOH, -CONH~, -CONHR', -CONR'R2,
-NH2, -NHR', -NR'R2, -NHCOR', -SH, SR', -F, -ONH2, -ONHR', -
ONR'R2, -NHOH, -NHOR', -NR20H, -NRZOR', substituted or unsubstituted
C,-C,o straight chain or branched alkyl, substituted or unsubstituted C~-C",
straight chain or branched alkenyl, substituted or unsubstituted C,-C",
straight
8
E W
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chain or branched alkynyl, substituted or unsubstituted C,-C,o straight chain
or branched alkoxy, substituted or unsubstituted Cz-C,o straight chain or
branched alkenyloxy, and substituted or unsubstituted C,-C,o straight chain or
branched alkynyloxy. The substituents for W groups are independently
halogen, cyano, amino, carboxy, ester, ether, carboxamide, hydroxy, or
mercapto. R' and RZ can be substituted or unsubstituted alkyl, alkenyl, or
alkynyl groups, where the substituents are independently halogen, cyano,
amino, carboxy, ester, ether, carboxamide, hydroxy, or mercapto.
D and E are residues which together form a phosphodiester or
phosphorothioate diester bond between adjacent nucleosides or nucleoside
analogues or together form an analogue of an internucleosidic bond.
B is hypoxanthin-9-yl, or a functional equivalent thereof, in X's~'; B
can be guanin-9-yl, hypoxanthin-9-yl or 7-deazaguanin-9-yl in Xs, X8, and
X'z; B can be adenin-9-yl, 2,6-diaminopurin-9-yl, purin-9-yl or 7-deazaa-
denin-9-yl in X6, X9, X'3, and X"; B can be uracil-1-yl, uracil-5-yl, thymin-
1-yl or 5-propynyluracil-1-yl in X4; B can be cytosin-1-yl, 5-methylcytosin-
1-yl or 5-propynylcytosin-1-yl in X3; and B can be adenin-9-yl, cytosin-1-yl,
guanin-9-yl, uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl, thymin-1-yl, 5-
methylcytosin-1-yl, 2,6-diaminopurin-9-yl, purin-9-yl, 7-deazaadenin-9-yl, 7-
deazaguanin-9-yl, 5-propynylcytosin-1-yl, 5-propynyluracil-1-yl, isoguanin-9-
yl, 2-aminopurin-9-yl, 6-methyluracil-1-yl, 4-thiouracil-1-yl, 2-pyrimidone-1-
yl, quinazoline-2,4-dione-1-yl, xanthin-9-yl, NZ-dimethyiguanin-9-yl, or a
functional equivalent thereof in X', X9", and X~'2. B of X's.' is preferably
hypoxanthin-9-yl or an analog where no hydrogen bond can form between
any group at the 2 position of the base and the 2-oxo group of C'6.'.
Preferably, B is not guanin-9-yl in X's.~.
B in X3, X°, Xs, X6, Xg, X9, X'2, X", and X'4 can also be a
functionally equivalent nucleobase within the context of the catalytic core of
a
hammerhead ribozyme. For example, C3, U4, Gs, and A6 in hammerhead
ribozymes form a structure closely resembling a uridine turn in a tRNA (Pley
et al., Nature 372:68-74 1994). Other groups of nucleotides can also form
uridine turns and so nucleotides X', X4, Xs, X6 may be replaced as a group
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with nucleotides or nucleotide analogues that have the potential to form a
structure resembling a uridine turn. Similarly, the sheared base pairs in the
catalytic core of hammerhead ribozymes have interactions that may be similar
to interactions of other non-canonical base pairs. Knowledge of the crystal
structure of the catalytic core of hammerhead ribozymes, combined with the
discovery that a Gerlach type hammerhead ribozyme in which a non-
canonical base pair has been replaced with an isostructural non-canonical base
pair is active, indicates that analogous isostructural base pair replacements
should be possible elsewhere in the catalytic core.
Definitions
As used herein, oligomer refers to oligomeric molecules composed of
subunits where the subunits can be of the same class (such as nucleotides) or
a mixture of classes (such as nucleotides and ethylene glycol). It is
preferred
that the disclosed oligomers be oligomeric sequences, non-nucleotide linkers,
or a combination of oligomeric sequences and non-nucleotide linkers. It is
more preferred that the disclosed oligomers be oligomeric sequences.
Oligomeric sequences are oligomeric molecules where each of the subunits
includes a nucleobase (that is, the base portion of a nucleotide or nucleotide
analogue) which can interact with other oligomeric sequences in a base-
specific manner. The hybridization of nucleic acid strands is a preferred
example of such base-specific interactions. Oligomeric sequences preferably
are comprised of nucleotides, nucleotide analogues, or both, or are
oligonucleotide analogues.
Non-nucleotide linkers can be any molecule, which is not an
oligomeric sequence, that can be covalently coupled to an oligomeric
sequence. Preferred non-nucleotide linkers are oligomeric molecules formed
of non-nucleotide subunits. Examples of such non-nucleotide linkers are
described by Letsinger and Wu, (J. Am. Chem. Soc. 117:7323-7328 (1995)),
Benseler et al., (J. Am. Chem. Soc. 115:8483-8484 (1993)) and Fu et al. ,
(J. Am. Chem. Soc. 116:4591-4598 (1994)). Preferred non-nucleotide
linkers, or subunits for non-nucleotide linkers, include substituted or
unsubstituted C,-C,o straight chain or branched alkyl, substituted or
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unsubstituted CZ-C,o straight chain or branched alkenyl, substituted or
unsubstituted CZ-C,o straight chain or branched alkynyl, substituted or
unsubstituted C,-C,o straight chain or branched alkoxy, substituted or
unsubstituted CZ C,o straight chain or branched alkenyloxy, and substituted or
unsubstituted C2-C,o straight chain or branched alkynyloxy. The substituents
for these preferred non-nucleotide linkers (or subunits) can be halogen,
cyano, amino, carboxy, ester, ether, carboxamide, hydroxy, or mercapto.
As used herein, nucleoside refers to adenosine, guanosine,
cytidine, uridine, 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-
deoxycytidine, or thymidine. A nucleoside analogue is a chemically modified
form of nucleoside containing a chemical modification at any position on the
base or sugar portion of the nucleoside. As used herein, the term nucleoside
analogue encompasses, for example, both nucleoside analogues based on
naturally occurring modified nucleosides, such as inosine and pseudouridine,
and nucleoside analogues having other modifications, such as modifications to
the 2' position of the sugar. As used herein, nucleotide refers to a phosphate
derivative of nucleosides as described above, and a nucleotide analogue is a
phosphate derivative of nucleoside analogues as described above. The
subunits of oligonucleotide analogues, such as peptide nucleic acids, are also
considered to be nucleotide analogues.
As used herein, a ribonucleotide is a nucleotide having a 2' hydroxyl
function. Analogously, a 2'-deoxyribonucleotide is a nucleotide having only
2' hydrogens. Thus, ribonucleotides and deoxyribonucleotides as used herein
refer to naturally occurring nucleotides having nucleoside components
adenosine, guanosine, cytidine, and uridine, or 2'-deoxyadenosine, 2'-
deoxyguanosine, 2'-deoxycytidine, and thymidine, respectively, without any
chemical modification. Ribonucleosides, deoxyribonucleosides,
ribonucleoside analogues and deoxyribonucleoside analogues are similarly
defined except that they lack the phosphate group, or an analogue of the
phosphate group, found in nucleotides and nucleotide analogues.
As used herein, oligonucleotide analogues are polymers of nucleic
acid-like material with nucleic acid-like properties, such as sequence
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dependent hybridization, that contain at one or more positions, a modification
away from a standard RNA or DNA nucleotide. A preferred example of an
oligonucleotide analogue is peptide nucleic acid.
As used herein, base pair refers to a pair of nucleotides or nucleotide
analogues which interact through one or more hydrogen bonds. The term
base pair is not limited to interactions generally characterized as Watson-
Crick base pairs, but includes non-canonical or sheared base pair interactions
(Topal and Fresco, Nature 263:285 (1976); Lomant and Fresco, Prog. Nucl.
Acid Res. Mol. Biol. 15:185 (1975)). Thus, nucleotides A'S~' and U'6~' form
a base pair in hammerhead ribozymes (see Figure I ) but the base pair is non-
canonical (see Figure 3).
The internucleosidic linkage between two nucleosides can be achieved
by phosphodiester bonds or by modified phospho bonds such as by
phosphorothioate groups or other bonds such as, for example, those described
in U.S. Pat. No. 5,334,711.
Flanking Elements Z, and Z4
The monomeric subunits of elements Z, and Z4 which flank the active
center (formed by elements ZZ and Z3) are preferably nucleotides and/or
nucleotide analogues. Elements Z, and Z4 are designed so that they
specifically interact, preferably by hybridization, with a given RNA substrate
and, together with the active center ZZ and Z3, form a structure (preferably a
structure resembling that of a hammerhead ribozyme) which specifically
cleaves the RNA substrate.
The subunits of elements Z, and Z4 can, on the one hand, be
ribonucleotides. However, it is preferred that the number of ribonucleotides
be as small as possible since the presence of ribonucleotides reduces the in
vivo stability of the oligomers. Elements Z, and Z4 (and also the active
center ZZ and Z3) preferably do not contain any ribonucleotides at the
positions containing pyrimidine nucleobases. Such positions preferably
contain nucleotide analogues.
The use of a large number of deoxyribonucleotides in elements Z, and
Z4 is also less preferred since undesired interactions with proteins can occur
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or an unintended RNase H-sensitive DNA-RNA hybrid could form. Thus,
elements Z, and Z4 each preferably contain (i) no ribonucleotides, and (2)
no sequences of more than 3 consecutive deoxyribonucleotides.
The subunits of elements Z, and Z4 are preferably nucleotides,
nucleotide analogues, or a combination. Preferably, the nucleotides and
nucleotide analogues in elements Z, and Z4 each have the structure
D
(I)
In structure (I) each B can be adenin-9-yl, cytosin-1-yl, guanin-9-yl,
uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl, thymin-1-yl, 5-methylcytosin-1-yl,
2,6-diaminopurin-9-yl, purin-9-yl, 7-deazaadenin-9-yl, 7-deazaguanin-9-yl, S-
propynylcytosin-1-yl, 5-propynyluracil-1-yl, isoguanin-9-yl, 2-aminopurin-9-
yl, 6-methyluracil-1-yl, 4-thiouracil-1-yl, 2-pyrimidone-1-yl, quinazoline-2,4-
dione-1-yi, xanthin-9-yl, NZ-dimethylguanin-9-yl or a functional equivalent
thereof;
Each V can be an O, S, NH, or CH, group.
Each W can be -H, -OH, -COOH, -CONH2, -CONHR', -CONR'R2,
-NH,, -NHR', -NR'Rz, -NHCOR', -SH, SR', -F, -ONHz, -ONHR', -
ONR'RZ, -NHOH, -NHOR', -NRZOH, -NRZOR', substituted or unsubstituted
C,-C,o straight chain or branched alkyl, substituted or unsubstituted CZ-C,o
straight chain or branched alkenyl, substituted or unsubstituted C~-C,~,
straight
chain or branched aikynyl, substituted or unsubstituted C,-C,o straight chain
or branched alkoxy, substituted or unsubstituted Cz-C,o straight chain or
branched alkenyloxy, and substituted or unsubstituted C~-C,o straight chain or
branched alkynyloxy. The substituents for W groups are independently
halogen, cyano, amino, carboxy, ester, ether, carboxamide, hydroxy, or
mercapto. R' and RZ can be substituted or unsubstituted alkyl, alkenyl, or
13
E IN
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alkynyi groups, where the substituents are independently halogen, cyano,
amino, carboxy, ester, ether, carboxamide, hydroxy, or mercapto.
D and E are residues which together form a phosphodiester or
phosphorothioate diester bond between adjacent nucleosides or nucleoside
analogues or together form an analogue of an internucleosidic bond.
For elements Z, and Z4 having nucleotide and/or nucleotide analogues
of structure (I), it is preferred that each W is substituted or unsubstituted
C,-
C,o straight chain or branched alkoxy, CZ-C,o straight chain or branched
aikenyloxy, or CZ-C,o straight chain or branched alkynyloxy.
In addition, the flanking elements Z, and Z4 can also contain nu-
cleotide analogues such as peptide nucleic acids (also referred to as peptidic
nucleic acids; see for example Nielsen et al., Science 254:1497-1500 (1991),
and Dueholm et al., J. Org. Chem. 59:5767-5773 (1994)). In this case the
coupling of individual subunits can, for example, be achieved by acid amide
bonds. Elements Z, and Z4, when based on peptide nucleic acids, can be
coupled to elements Z~ and Z3, based on nucleotides or nucleotide analogues,
using either suitable linkers (see, for example, Petersen et al. , BioMed.
Chem. Lett. 5:1119-1121 (1995)) or direct coupling (Bergmann et al.,
Tetrahedron Lett. 36:6823-6826 (1995)). Where elements Z, and Z4 contain
a combination of nucleotides {and/or nucleotide analogues) and peptide
nucleic acid, similar linkages can be used to couple the different parts.
The subunits of the flanking elements Z, and Z4 contain nucleobases
or nucleobase analogues which can hybridize or interact with nucleobases that
occur naturally in RNA molecules. The nucleobases are preferably selected
from naturally occurring bases (that is, adenine, guanine, cytosine, thymine
and uracil) as well as nucleobase analogues, such as 2,6-diaminopurine,
hypoxanthine, 5-methylcytosine, pseudouracil, 5-propynyluracil, and S-
propynylcytosine, which enable a specific binding to the target RNA.
A strong and sequence-specific interaction (that is, a more stable
hybrid between the RNA substrate and the oligomer) between the RNA
substrate and elements Z, and Z4 is preferred. For this purpose, it is
preferred that the following nucleobase analogues be used in oligomeric
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sequences of elements Z, and Z4 in place of the standard nucleobases: 2,6- .
diaminopurine instead of adenine; thymine or 5-propynyluracil instead of
uracil; and 5-methylcytosine or 5-propynylcytosine instead of cytosine. 2-
Amino-2'-O-alkyladenosines are also preferred (Lamm et al. , Nucleic Acids
Res. 19:3193-3198 (1991)). Furthermore, aromatic systems can be linked to
positions 4 and 5 of uracil to produce nucleobase analogues such as
phenoxazine, which can improve the stability of the double-strand (Lin et al.
,
J. Am. Chem. Soc. 117:3873-3874 (1995)).
Preferred RNA substrates for cleavage by the disclosed compositions
have the structure
5~-Z4~_ye.'-Xm_Z~ ~-3',
where Z'' and Z4' interact with Z, and Z4, respectively, where C'6' is
cytidine, and where X" is adenosine, guanosine, cytidine, or uridine.
Cleavage occurs 3' of X". Preferably, X" is adenosine, cytidine, or uridine,
more preferably X" is adenosine or cytidine, and mast preferably X" is
adenosine. Preferably, X'6'z (that is, the 3' nucleoside in Z4') is adenosine
or
guanosine. The target sites in substrates which can be cleaved by the
disclosed compositions are distinct from target sites for previous hammerhead
ribozymes since previous hammerhead ribozymes require a uridine in position
16.1 of the substrate.
Position N'6.z, which is the 3' most position present in Z4', can be
either a guanosine, adenosine, cytidine, or uridine. It is preferred that
N'b.z
is either a guanosine, adenosine, or cytidine. It is more preferred that N'b.z
is either guanosine or adenosine. It is most preferred that N'6'z is
guanosine.
Preferred substrates for cleavage by the disclosed compounds are those where
N16.2 is guanosine, adenosine, or cytidine and X" is adenosine. More
preferred substrates for cleavage by the disclosed compounds are those where
N'6~z is guanosine or adenosine and X" is adenosine. Most preferred
substrates for cleavage by the disclosed compounds are those where N'b~z is
guanosine and X" is adenosine.
Flanking elements Z, and Z4 preferably contain, independently of each
other, from 3 to 40, and more preferably from 5 to 10, nucleotides or
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nucleotide analogues. It is preferred that Z, and Z,' interact to form a stem
.
of at least three base pairs, and that Z4 and Z4' interact to form a stem of
at
least three base pairs. It is more preferred that these stems are adjacent to
Z,
and Z3, respectively. It is most preferable that Z, and Z,' interact to form a
stem of more than three base pairs, and that Z4 and Z4' interact to form a
stem of more than three base pairs.
Preferred RNA substrates are those that have little inhibitory
secondary structure associated with the target region of the RNA. There are
a number of ways to determine which regions of an RNA molecule contain
secondary structure, and would therefore be less preferred, and which regions
of an RNA molecule have little secondary structure, and therefore, would be
more preferred. Preferred methods for determining regions of single-
stranded RNA are those that map the single-stranded regions of RNA by
selectively reacting with or recognizing these regions. There are many
chemicals (dimenthyl sulfate (DMS), diethylpyrocarbonate {DEPC), cathoxal
(CMCT), carbodiimides) which react with nitrogens at the Watson-Crick face
of nucleotides. Nucleotides involved in Watson-Crick base pairing show less
reactivity with these chemicals than nucleotides which are not. Enzymatic
reactions (using reverse transcriptase, RNase T1, cobra venom nuclease,
nuclease S 1, nuclease V 1 ) with chemically modified RNA create shortened
oligonucleotides whose length is dependant on the base where the chemical
reaction occurred. Since chemical reactions occur preferentially at the single
stranded regions of the RNA, these techniques indicate where the secondary
structure of the RNA is. For example, methods such as dimethyl sulfate and
reverse transcription mapping indicate regions of double-stranded RNA.
Reverse transcriptase under the appropriate conditions is unable to process
through regions of double-stranded RNA, and therefore, there are abortive
transcripts which when analyzed by polyacrylamide gel electrophoresis
(PAGE) indicate where in the RNA regions of strong secondary structure
exist. Examples of these methods are described by Kumar et al.,
Biochemistry 33(2):583-592 (1994), Mandiyan and Boublik, Nucleic Acids
Res. 18(23):7055-7062 (1990}, Bernal and Garcia-Arenal, RNA 3(9):1052-
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1067 (1997), and Ehresmann et al., Nucleic Acids Res. 15(22):9109-9128
( 1987).
Another method which assesses which regions of RNA are single-
stranded is RNase H mapping. In this method, short, random DNA
oligonucleotides are synthesized and mixed with the target RNA. Regions of
easy accessibility are hybridized with the short DNA molecules. The DNA-
RNA hybrid regions are then cleaved by RNase H. The labeled RNA
molecule can then be analyzed by PAGE. By comparing the cleaved
molecules with a sequencing ladder, the regions of single-stranded DNA can
be inferred. Examples of this method are described by Ho et al. , Nature
Biotechnology 16:59-63 (1998), and in U.S. Patent No. 5,525,468 to
McSwiggen.
In vitro selection experiments (Szostak, TIBS 19:89-93 (1992)) can
also be performed to determine the accessible single-stranded regions of
RNA. For example, the target RNA can be mixed with random DNA
oligonucleotides that contain primer binding regions which can be used for
PCR amplification. Amplification and reselection in an iterative manner will
allow for enrichment of those DNA sequences which are capable of binding
the RNA. This identifies the regions of the RNA which are accessible for
oligonucleotide hybridization. The selection or enrichment step can be any
size selection or double-stranded nucleic acid separation technique. For
example, Sephadex column chromotagraphy will separate the large, bound
DNA:RNA complexes and the small unbound DNA molecules, or
nitrocellulose filtration will retain the bound RNA while the unbound DNA
molecules will flow through.
There are also a number of methods for optimizing the oligomers for
a given substrate. For example, position N' of the oligomers, which has no
specific sequence requirements, can be changed to help minimize the
' possibility of unwanted secondary structure in the oligomers designed for a
given target sequence. Also specific base modifications, such as 7-deaza-
guanosine or 7-deaza-adenosine, can be utilized in regions having a number
of guanosines or adenosines to prevent unwanted purine:purine interactions.
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Similar substitutions can be accomplished by introducing inosine into
guanosine rich regions, that may be present for example in regions Z' or Z4
of the catalytic oligomer.
Catalytic Core
Elements ZZ and Z3 are considered to form the catalytic core of the
disclosed compositions, and are preferably made up of nucleotide analogues
and a small number of ribonucleotides. In elements ZZ and Z3 it is preferred
that each W (in structure (I)) is C'-C5 straight chain or branched alkyl, CZ-
CS
straight chain or branched alkenyl, CZ-CS straight chain or branched alkynyl,
C,-CS straight chain or branched alkoxy, CZ-CS straight chain or branched
alkenyloxy, and CZ-C5 straight chain or branched CZ-CS alkynyloxy. It is also
preferred that in each X', X°, X' and X"'Z, W is NH2, OH-substituted C'-
C4
alkyl, OH-substituted CZ-C4 alkenyl, OH-substituted C'-C4 alkoxy or OH-
substituted C~-C4 alkenyloxy. It is more preferred that in each X3, X", X'
and X"'2, W is NHZ, methoxy, 2-hydroxyethoxy, allyloxy or allyl. It is also
preferred that in X'2, W is -H or -OH. It is also preferred that in each X'3
and X'4, W is C1-C4 alkyl, CZ-CQ alkenyl, C'-C4 alkoxy, CZ-C4 alkenyloxy,
OH-substituted C,-C4 alkyl, OH-substituted Cz-C4 alkenyl, OH-substituted C'-
C4 aikoxy, or OH-substituted CZ C4 alkenyloxy. It is more preferred that in
each X'3 and X'4, W is methoxy, 2-hydroxyethoxy or allyloxy.
The subunits in elements Z~ and Z3 are preferably nucleotide
analogues which can only hybridize weakly with ribonucleotides. Examples
of such subunits are nucleotide analogues that contain a substituted or
unsubstituted alkyl, alkenyl, alkynyl, alkoxy, alkenyloxy or alkynyloxy
group, with preferably 1 to 5 carbon atoms, at the 2' position of ribose.
Preferred nucleobases which can be used in elements Z, and Z3 for this
purpose are adenin-9-yl, purin-9-yl, uracil-1-yl, cytosin-1-yl, guanin-9-yl
and
hypoxanthin-9-yl.
The following nucleotides and nucleotide analogues are preferred for
element ZZ (referring to components of structure (I)):
Position X3: B = cytosin-1-yl, V = O, W = allyloxy; B = cytosin-1-
yl, V = O, W = allyl;
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Position X": B = uracil-1-yl, V = O, W = allyloxy; B = uracil-1-yl,
V = O, W = allyl; B = uracil-1-yl, V = O, W = OH;
Position X5: B = guanin-9-yl, V = O, W = amino; B = guanin-9-yl,
V = O, W = OH;
Position X6: B = adenin-9-yl, V = O, W = H; B = purin-9-yl, V =
O, W = OH; B = adenin-9-yl, V = O, W = OH;
Position X': B = uracil-1-yl, V = O, W = allyloxy; B = cytosin-1-
yl, V = O, W = allyl;
Position X8: B = guanin-9-yl, V = O, W = amino; B =
hypoxanthin-9-yl, V = O, W = OH; B = guanin-9-yl, V = O, W = OH;
Position X9: B = adenin-9-yl, V = O, W = H; B = purin-9-yl, V =
O, W = OH; B = adenin-9-yl, V = O, W = allyloxy.
The following nucleotides and nucleotide analogues are preferred for
element Z, (referring to components of structure (I)):
Position X'2: B = guanin-9-yl, V = O, W = H; B = 7-deazaguanin-
9-yl, V = O, W = OH; B = guanin-9-yl, V = O, W = OH;
Position X'3: B = adenin-9-yl, V = O, W = allyloxy; or B =
adenin-9-yl, V = O, W = 2-hydroxyethoxy; B = purin-9-yl, V = O, W =
allyloxy;
Position X'4: B = adenin-9-yl, V = O, W = allyloxy; B = purin-9-
yl, V = O, W = OH; B = adenin-9-yl, V = O, W = 2-hydroxyethoxy; B
= purin-9-yl, V = O, W = allyloxy;
Position X'S~': B = hypoxanthin-9-yl or a functional equivalent
thereof, V = O, W = OH.
Elements ZZ and Z3 interact in a way that allows for the formation of
a catalytic structure. In preferred compositions ZZ and Z3 interact in a way
that allows for the formation of a catalytic structure resembling a
hammerhead catalytic structure. One way ZZ and Z3 can interact to form a
catalytic structure is through the interaction of the nucleotides and/or
nucleotide analogues making up ZZ and Z3. The disclosed compositions have
an RNA cleaving activity independent of RNase H. That is, the disclosed
compositions are able to cause cleavage of an RNA substrate without
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involving RNase H. Although the disclosed compositions may also be
capable of promoting cleavage of RNA by RNase H, it is preferred that they
do not.
Desired interaction between Z, and Z3 is preferably enhanced by
coupling elements to the 3'-end of ZZ and/or the 5'-end of Z3. A single
element (referred to herein as ZS) can be used in this way to covaiently
couple elements ZZ and Z3. The structure of such a form of the disclosed
compositions would be 5'-Z,-Z~-ZS-Z3-Z4-3'. Separate elements (referred to
herein as Z6 and Z,) can be coupled to ZZ and Z3 which preferably interact
(non-covalently) to stabilize or otherwise enhance the interaction of elements
ZZ and Z3. Component (a) of a composition of this form would have the
structure 5'-Z,-Zz-Z~-3', and component (b) would have the structure 5'-Z,-
Z3-Z4-3' .
It is preferred that elements Z5, Z6 and Z~ are oligomeric sequences,
non-nucleotide linkers, or a combination of oligomeric sequences and non-
nucleotide linkers. It is more preferred that elements Z5, Z6 and Z~ are
oligomeric sequences. It is most preferred that these oligomeric sequences
interact to form an intramolecular stem (in the case of ZS) or an
intermolecular stem (in the case of Z6 and Z~). Such stems preferably
contain from 2 to 30 base pairs, and are preferably continuous (that is,
lacking unpaired bases). Elements Z5, Z6 and Z~ preferably are comprised of
nucleotides, nucleotide analogues, or both, or are oligonucleotide analogues.
It is preferred that ZS interacts with itself in such a way as to stabilize
the
interactions between ZZ and Z3. Similarly, it is preferred that Z6 interact
with Z~ in such a way as to stabilize the interactions between ZZ and Z3. It
is
preferred that elements are oligomeric sequences made up of nucleotides
and/or nucleotide analogues, oligonucleotide analogues, or a combination,
which are able to hybridize with each other.
Element Z5 can serve as a covalent linker coupling the 3' end of ZZ to
the 5' end of Z3. Element ZS is preferably made up of either non-nucleotide
molecules such as polyethylene glycol, or oligomeric sequences, including
nucleotides, nucleotide analogues and oligonucleotide analogues, or a
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combination of nucleotides, nucleotide analogues and oligonucleotide
analogues. A preferred form of element Z5 is made up of nucleotides,
nucleotide analogues, oligonucleotide analogues, or a combination of
nucleotides and nucleotide analogues which are able to interact
intramolecularly to form a stem-loop structure. A preferred stem-loop
structure for element Z5 is one containing from 2 to 30 base pairs.
A preferred embodiment of the disclosed compositions is 5'-Z,-ZZ-ZS-
Z3-Z4-3' where all of the nucleotides are either 2'-O-allyl-ribonucleotides or
2'-O-methyl-ribonucleotides except for positions X4, X5, X6, Xg, X'z, X's.~
which are ribonucleotides unmodified at the 2' position (W = OH).
Another preferred embodiment of the disclosed compositions is 5'-Z,-
ZZ-ZS-Z3-Z4-3' where all of the nucleotides are either 2'-O-allyl-
ribonucleotides or 2'-O-methyl-ribonucleotides except far positions X5, X6,
X8, X'Z, X'5.' which are ribonucleotides unmodified at the 2' position (W =
OH).
Another preferred embodiment of the disclosed compositions is a
composition made up of components (a) and (b) as described above where
component (a) is 5'-Z,-ZZ-Z6 -3' and component (b) is 5'-Z~-Z3-Z4-3', where
all of the nucleotides are either 2'-O-allyl-ribonucleotides or 2'-O-methyl-
ribonucleotides except for positions X', X5, X6, Xx, X'2, X'S.' which are
ribonucleotides unmodified at the 2' position (W = OH).
Another preferred embodiment of the disclosed compositions is a
composition made up of components (a) and (b) as described above where
component (a) is 5'-Z'-ZZ-Z6 -3' and component (b) is 5'-Z,-Z3-Z4-3', where
all of the nucleotides are either 2'-O-allyl-ribonucleotides or 2'-O-methyl-
ribonucleotides except for positions X5, Xb, Xg, X'2, X'5.' which are
ribonucleotides unmodified at the 2' position (W = OH).
A preferred form of the disclosed composition is one in which there is
a G added to the 3' end of Z2. Taira and co-workers (Amontov and Taira, J.
Am. Chem. Soc. 118:1624-1628 (1996)) have shown that the stacking energy
gained from a guanosine juxtaposed to R9 of a hammerhead-like ribozyme
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stabilizes the formation of a catalytic structure. Thus, it is preferred that
the
5' nucleotide of Z6 is G.
The 3' end of the disclosed compositions can be protected against
degradation by exonucleases by, for example, using a nucleotide analogue
that is modified at the 3' position of the ribose sugar (for example, by
including a substituted or unsubstituted alkyl, alkoxy, alkenyl, alkenyloxy,
al-
kynyi or alkynyloxy group as defined above). The disclosed compositions
can also be stabilized against degradation at the 3' end by exonucleases by
including a 3'-3'-linked dinucleotide structure (Ortigao et al., Antisense
Research and Development 2:129-14b (1992)) and/or two modified phospho
bonds, such as two phosphorothioate bonds.
The disclosed compositions can also be linked to a prosthetic group in
order to improve their cellular uptake and/or to enable a specific cellular
localization. Examples of such prosthetic groups are polyamino acids (for
example, polylysine), lipids, hormones or peptides. These prosthetic groups
are usually linked via the 3' or 5' end of the oligomer either directly or by
means of suitable linkers (for example, linkers based on 6-aminohexanol or
6-mercaptohexanol). These linkers are commercially available and
techniques suitable for linking prosthetic groups to the oligomer are known to
a person skilled in the art.
Increasing the rate of hybridization can be important for the biological
activity of the disclosed compositions since in this way it is possible to
achieve a higher activity at low concentrations of the composition. This is
important for short-Lived RNA substrates or RNA substrates that occur less
often. A substantial acceleration of the hybridization can be achieved by, for
example, coupling positively charged peptides (containing, for example,
several lysine residues) to the end of an oligonucleotide (Corey J. Am. Chem.
Soc. 117:9373-9374 (1995)). The disclosed compositions can be simply
modified in this manner using the linkers described above. Alternatively, the
rate of hybridization can also be increased by incorporation of subunits which
contain sperminyl residues (Schmid and Behr, Tetrahedron Lett. 36:1447-
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1450 (1995)). Such modifications of the disclosed compositions also improve
the ability to bind to RNA substrates having secondary structures.
Synthesis of Oligomers
The disclosed compositions can be synthesized using any suitable
method. Many synthesis methods are known. The following techniques are
preferred for synthesis of the disclosed compositions. 2'-O-Allyl modified
oligomers that contain residual purine ribonucleotides, and bearing a suitable
3'-terminus such as an inverted thymidine residue {Ortigao et al., Antisense
Research and Development 2:129-146 (1992)) or two phosphorothioate
linkages at the 3'-terminus to prevent eventual degradation by 3'-
exonucleases, can be synthesized by solid phase a-cyanoethyl
phosphoramidite chemistry (Sinha et al. , Nucleic Acids Res. 12:4539-4557
(1984)) on any commercially available DNA/RNA synthesizer. A preferred
method is the 2'-O-tent-butyldimethylsilyi (TBDMS) protection strategy for
the ribonucleotides (Usman et al. , J. Am. Chem. Soc. 109:7845-7854
(1987)), and all the required 3'-O-phosphoramidites are commercially
available. In addition, the use of aminomethylpolystyrene is preferred as the
support material due to its advantageous properties (McCollum and Andrus
Tetrahedron Levers 32:4069-4072 (1991)). Fluorescein can be added to the
5'-end of a substrate RNA during the synthesis by using commercially
available fluorescein phosphoramidites. In general, a desired oligomer can
be synthesized using a standard RNA cycle. Upon completion of the
assembly, all base labile protecting groups are removed by an 8 hour
treatment at SS °C with concentrated aqueous ammonia/ethanol (3:1 v/v)
in a
sealed vial. The ethanol suppresses premature removal of the 2'-O-TBDMS
groups which would otherwise lead to appreciable strand cleavage at the
resulting ribonucleotide positions under the basic conditions of the
deprotection (Usman et al. , J. Am. Chem. Soc. 109:7845-7854 ( 1987)).
After lyophilization the TBDMS protected oligomer is treated with a mixture
of triethylamine trihydrofluoride/triethylamine/N-methylpyrrolidinone for 2
hours at 60°C to afford fast and efficient removal of the silyl
protecting
groups under neutral conditions (Wincott et al. , Nucleic Acids Res. 23:2677-
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2684 (1995)). The fully deprotected oligomer can then be precipitated with
butanol according to the procedure of Cathala and Brunel (Nucleic Acids Res.
18:201 (1990)). Purification can be performed either by denaturing
polyacrylamide gel electrophoresis or by a combination of ion-exchange
HPLC (Sproat et al., Nucleosides and Nucleotides 14:255-273 (1995)) and
reversed phase HPLC. For use in cells, it is preferred that synthesized
oligomers be converted to their sodium salts by precipitation with sodium
perchlorate in acetone. Traces of residual salts are then preferably removed
using small disposable gel filtration columns that are commercially available.
As a final step it is preferred that the authenticity of the isolated
oligomers is
checked by matrix assisted laser desorption mass spectrometry (Pieles et al. ,
Nucleic Acids Res. 21:3191-3196 (1993)) and by nucleoside base composition
analysis. In addition, a functional cleavage test with the oligomer on the
corresponding chemically synthesized short oligoribonucleotide substrate is
also preferred.
Cleavage of RNA Substrates
The disclosed compositions have a very high in vivo activity since the
RNA cleavage is promoted by protein factors that are present in the nucleus
or cytoplasm of the cell. Examples of such protein factors which can
increase the activity of hammerhead ribozymes are, for example, the
nucleocapsid protein NCp7 of HIV 1 (Mullet et al. , J. Mol. Biol. 242:422-
429 (1994)) and the heterogeneous nuclear ribonucleoprotein A1 (Heidenreich
et al., Nucleic Acids Res. 23:2223-2228 (1995)). Thus, long RNA transcripts
can be cleaved efficiently within the cell by the disclosed compositions.
The disclosed compositions can be used in pharmaceutical
compositions that contain one or several oligomers as the active substance,
and, optionally, pharmaceutically acceptable auxiliary substances, additives
and carriers. Such pharmaceutical compositions are suitable for the produc-
tion of an agent to specifically inactivate the expression of genes in eukary-
otes, prokaryotes and viruses, especially of human genes such as tumor genes
or viral genes or RNA molecules in a cell. Further areas of application are
the inactivation of the expression of plant genes or insect genes. Thus, the
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disclosed compositions can be used as drugs for humans and animals as well
as a pesticide for plants.
A variety of methods are available for delivering the disclosed
compositions to cells. For example, in general, the disclosed compositions
can be incorporated within or on microparticles. As used herein,
microparticles include liposomes, virosomes, microspheres and microcapsules
formed of synthetic and/or natural polymers. Methods for making
microcapsules and microspheres are known to those skilled in the art and
include solvent evaporation, solvent casting, spray drying and solvent
extension. Examples of useful polymers which can be incorporated into
various microparticles include polysaccharides, polyanhydrides,
polyorthoesters, polyhydroxides and proteins and peptides.
Liposomes can be produced by standard methods such as those
reported by Kim et al., Biochim. Biophys. Acta, 728:339-348 (1983); Liu et
al., Biochim. Biophys. Acta, 1104:95-101 (1992); and Lee et al., Biochim.
Biophys. Acta., 1103:185-197 (1992); Wang et al., Biochem., 28:9508-9514
(1989)). Such methods have been used to deliver nucleic acid molecules to
the nucleus and cytoplasm of cells of the MOLT-3 leukemia cell line (Thierry
and Dritschilo, Nucl. Acids Res., 20:5691-5698 (1992)). Alternatively, the
disclosed compositions can be incorporated within microparticles, or bound to
the outside of the microparticles, either ionically or covalently.
Cationic liposomes or microcapsules are microparticles that are
particularly useful far delivering negatively charged compounds such as the
disclosed compounds, which can bind ionically to the positively charged outer
surface of these liposomes. Various cationic liposomes have previously been
shown to be very effective at delivering nucleic acids or nucleic acid-protein
complexes to cells both in vitro and in vivo, as reported by Felgner et al. ,
Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987); Felgner, Advanced Drug
Delivery Reviews, S:i63-187 (1990); Clarenc et al., Anti-Cancer Drug
Design, 8:81-94 (/993). Cationic liposomes or microcapsules can be
prepared using mixtures including one or more lipids containing a cationic
side group in a sufficient quantity such that the liposomes or microcapsules
CA 02295207 1999-12-15
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formed from the mixture possess a net positive charge which will sonically
bind negatively charged compounds. Examples of positively charged lipids
that may be used to produce cationic liposomes include the aminolipid
dioleoyl phosphatidyl ethanolamine (PE), which possesses a positively
charged primary amino head group; phosphatidylcholine (PC), which possess
positively charged head groups that are not primary amines; and N[1-(2,3-
dioleyloxy)propyl)-N,N,N-triethylammonium ("DOTMA," see Felgner et al.,
Proc. Natl. Acad. Sci USA, 84:7413-7417 (1987); Felgner et al., Nature,
337:387-388 (1989); Felgner, Advanced Drug Delivery Reviews, 5:163-187
(1990)).
A preferred form of microparticle for delivery of the disclosed
compositions are heme-bearing microparticles. In these microparticles, heme
is intercalated into or covalently conjugated to the outer surface of the
microparticles. Heme-bearing microparticles offer an advantage in that since
they are preferentially bound and taken up by cells that express the heme
receptor, such as hepatocytes, the amount of drug required for an effective
dose is significantly reduced. Such targeted delivery may also reduce
systemic side effects that can arise from using relatively high drug
concentrations in non-targeted delivery methods. Preferred lipids for forming
heme-bearing microparticles are 1,2-dioleoyloxy-3-(trimethylammonium)
propane (DOTAP) and dioleoyl phosphatidyl ethanolamine (DOPE). The
production and use of heme-bearing microparticles are described in PCT
application WO 95/27480 by Innovir.
The disclosed compositions can also be encapsulated by or coated on
cationic liposomes which can be injected intravenously into a mammal. This
system has been used to introduce DNA into the cells of multiple tissues of
adult mice, including endothelium and bone marrow, where hematopoietic
cells reside (see, for example, Zhu et al., Science, 261:209-211 (1993)).
Liposomes containing the disclosed compositions can be administered
systemically, for example, by intravenous or intraperitoneal administration,
in
an amount effective for delivery of the disclosed compositions to targeted
cells. Other possible routes include trans-dermal or oral, when used in
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conjunction with appropriate microparticles. Generally, the total amount of
the liposome-associated oligomer administered to an individual will be less
than the amount of the unassociated oligomer that must be administered for
the same desired or intended effect.
Compositions including various polymers such as the polylactic acid
and polyglycolic acid copolymers, polyethylene, and polyorthoesters and the
disclosed compositions can be delivered locally to the appropriate cells by
using a catheter or syringe. Other means of delivering such compositions
locally to cells include using infusion pumps (for example, from Alza
Corporation, Palo Alto, California) or incorporating the compositions into
polymeric implants (see, for example, Johnson and Lloyd-Jones, eds., Drug
Delivery Systems (Chichester, England: Ellis Horwood Ltd. , 1987), which
can effect a sustained release of the therapeutic compositions to the
immediate area of the implant.
For therapeutic applications the active substance is preferably
administered at a concentration of 0.01 to 10,000 ~,g/kg body weight, more
preferably of 0.1 to 1000 ~,g/kg body weight. The administration can, for
example, be carned out by injection, inhalation (for example as an aerosol),
as a spray, orally (for example as tablets, capsules, coated tablets etc.),
topically or rectally (for example as suppositories).
The disclosed compositions can be used in a method for the specific
inactivation of the expression of genes in which an active concentration of
the
composition is taken up into a cell so that the composition specifically
cleaves
a predetermined RNA molecule which is present in the cell, the cleavage
preferably occurring catalytically. Similar compositions, which are described
in U.S. Patent No. 5,334,711, have been used successfully in mice to
inactivate a gene (Lyngstadaas et al., EMBO J. 14:5224-5229 (I995)). This
process can be carried out in vitro on cell cultures as well as in vivo on
living
organisms (prokaryotes or eukaryotes such as humans, animals or plants).
The disclosed compositions can also be used as RNA restriction
enzymes to cleave RNA molecules (in, for example, cell free in vitro
reactions). The disclosed compositions can also be used in a reagent kit for
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the restriction cleavage of RNA molecules which contains, for example, an
oligomer and suitable buffer substances. In this case the oligomer and the
buffer substances can be present in the form of solutions, suspensions or
solids such as powders or lyophilisates. The reagents can be present
together, separated from one another or optionally also on a suitable carrier.
The disclosed compositions can also be used as a diagnostic agent or to
identify the function of unknown genes.
The present invention will be further understood by reference to the
following non-limiting examples.
Examples
Example 1: Cleavage reactions which indicate that an inosine
substitution at position 15.1 can effectively cleave
N16.2~16.IH17~
A set of 12 substrates was synthesized which covered each
permutation of the N'6.2C'6.1H1' motif where H" is not guanosine. The
oligomers and the corresponding substrates used in the cleavage assays are
shown in Table 1. Each of the substrates was labeled with fluorescein at the
5' end and an inverted thymidine cap was used on the 3'-end. A set of four
catalytic oligomers was synthesized, providing an appropriately matched
catalytic oligomer for each of the substrates. Each of these catalytic
oligomers had an inosine at position 15.1. A control substrate and catalytic
oligomer were also synthesized in which there was a U at position 16.1 of
the substrate and an A at position 15.1 of the catalytic oligomer.
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Table 1
Ni6.zNi6.iHm
Triplet Substrate sequence
ACC Fl-GAAUACCGGUCGC*T (SEQ ID N0:4)
ACA Fl-GAAUACAGGUCGC*T (SEQ ID NO:S)
ACU FI-GAAUACUGGUCGC*T (SEQ ID N0:6)
GCC FI-GAAUGCCGGUCGC*T (SEQ ID N0:7)
GCA Fl-GAAUGCAGGUCGC*T (SEQ ID N0:8)
GCU FI-GAAUGCUGGUCGC*T (SEQ ID N0:9)
CCC FI-GAAUCCCGGUCGC*T (SEQ ID NO:10)
CCA FI-GAAUCCAGGUCGC*T (SEQ ID NO:11)
CCU Fl-GAAUCCUGGUCGC*T (SEQ ID N0:12)
25
UCC FI-GAAUUCCGGUCGC*T (SEQ ID N0:13)
UCA Fl-GAAUUCAGGUCGC*T (SEQ ID N0:14)
UCU Fl-GAAUUCUGGUCGC*T (SEQ ID NO:15)
GUC Fl-GAAUGUCGGUCGC*T (SEQ ID N0:16)
Targeted
triplet Catalytic oligomer sequence
ACH gcgacccuGAuGaggccgugaggccGaaIuauuc*T(SEQ ID N0:17)
GCH gcgacccuGAuGaggccgugaggccGaaIcauuc*T(SEQ ID N0:18)
CCH gcgacccuGAuGaggccgugaggccGaaIgauuc*T(SEQ ID N0:19)
UCH gcgacccuGAuGaggccgugaggccGaaIaauuc*T(SEQ ID N0:20)
GUC gcgacccuGAuGaggccgugaggccGaaAcauuc*T (SEQ ID N0:21)
FI = Fluorescein label
*T = 3'-3' inverted thymidine
A, C, G, I, U = ribonucleotides (I is inosine)
a, c, g, a = 2'-O-allyl-ribonucleotides
The above substrates and catalytic oligomers were used in cleavage
reactions to determine the ability of an inosine at position 15.1 to overcome
the requirement of a U at position 16.1 for cleavage. All of the reactions
were performed using the following protocol. The reactions were typically
done in 100 ~,1 and they contained distilled, autoclaved HZO, 10 mM MgClz,
10 mM Tris-HCl pH 7.4, 5 ~,M ribozyme, and 0.25 ~,M substrate. The
catalytic oligomer, substrate, and buffer were added together and heated to
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95 °C for 5 minutes. After cooling to room temperature over 5 minutes
the
reactions were brought to 10 mM MgClz , mixed, and placed at 37°C. 10
~cL aliquots were removed at specific time intervals (10, 30, 60, and 120
minutes) and added to 3 ~cl of loading buffer (95 % formamide, 100 mM
EDTA pH 8.0, 0.05 % bromophenol blue) to quench the reaction. Samples
were analyzed by 20 % polyacrylamide gel electrophoresis. Gels were
analyzed on a Molecular Dynamics Fluorescence Imager. The results of
cleavage reactions of this type, using the substrates and catalytic oligomers
shown in Table 1, are shown in Table 2.
Table 2
N16.2 N~6.~ Hm After
Triplet mixing 10 30 60 120
I~s.~ Uls.z Catalytic
oligomer
ACC 4.4 28.2 58.1 91.5 91.5
ACA 7.7 71.8 84.7 93.1 94.8
ACU 1.8 58.7 70.5
I~s.~ C~s.z Catalytic
oligomer
GCC 1.62 39.6 59.9 82.0 87.0
GCA 13.7 65.3 78.7 89.7 93.1
GCU -- 64.3 74.8
ys.~ G~s.z Catalytic
oligomer
CCC -- 34.33 45.38
CCA 1.1 18.8 45.5 70.8 80.63
CCU 2.0 28.4 36.7
I~s.~ A~s.z Catalytic
oligomer
UCC ' 6.8 57.0 64.7
UCA 1.6 39.6 60.8
UCU 3.3 41.1 53.1
A~s.~ C~s.z Catalytic
oligomer
GUC 1.6 38.5 66.5 93.5
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The numbers represent the percentage of substrate -cleaved at the
indicated time point (which were at 0, 10, 30, 60, and 120 minutes after
starting the reaction). The results indicate that substrates with a C at
position
16.1 are able to be cleaved by catalytic oligomers containing an I at position
15.1. While there are differences between the various substrates at the 120
minute time point, the data show that a substrate with a C at position 16.1 in
conjunction with a catalytic oligomer with an I at position 15.1 is able to
effectively cleave in all backgrounds, indicating that the substitution of an
I at
position 15.1 does in fact allow for the cleavage of any appropriate substrate
containing a Ni6.zC'6.~H~7 site.
Initial rates of cleavage of the twelve substrates having C'6', and the
control substrate having U'6~', by the corresponding catalytic oligomers (all
shown in Table 1) were determined using single turnover kinetics. Single
turnover kinetics were assessed by mixing 2.5 ~,l of a 100 ~,M ribozyme
solution, 2.5 ~,1 of a 10 ~M solution of 5' fluorescein labeled substrate, and
10 ~,1 of a 100 mM Tris-HCl pH 7.4 solution. The mixture was diluted to a
final volume of 90 ~,1, heated to 95°C for 5 minutes, and cooled to
37°C.
The reaction was started by adding 10 ~,1 of a 100 mM MgCl2 solution. The
final concentrations of the reaction components were 250 nM substrate, 2.5
~.mol ribozyme, and 10 mM MgCl2. Ten microliter samples were removed
at various times and mixed with 10 ~.l of a 100 mM EDTA, bromphenol blue
solution to stop the reaction. Cleavage products were separated from
unreacted substrate by PAGE and were quantitated on a Molecular Dynamics
Fluorescence Imager.
The data, measured in fraction of substrate cleaved versus time, were
fitted to the equation:
frac[P] = Ho(1-e kZ')/So
. as described by Jankowsky and Schwenzer, Nucl. Acids Res. 24:433 (1996).
The calculated values of k, for the various ribozymes are shown in Table 3.
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Table 3
S
N~b.2N~6.~Hm
Triplet
gcgacccuGAuGaggccgugaggccGaaIuauuc*T (SEQ ID N0:17)
k2 (min') Substrate sequence
ACC 0.07 FI-GAAUACCGGUCGC*T (SEQ ID N0:4}
ACA 0.36 Fl-GAAUACAGGUCGC*T (SEQ ID NO:S)
ACU 0.026 FI-GAAUACUGGUCGC*T (SEQ ID N0:6)
gcgacccuGAuGaggccgugaggccGaaIcauuc*T (SEQ ID N0:18)
kZ (miri') Substrate sequence
GCC 0.12 Fl-GAAUGCCGGUCGC*T (SEQ ID N0:7)
GCA 0.48 Fl-GAAUGCAGGUCGC*T (SEQ ID N0:8)
GCU 0.05 FI-GAAUGCUGGUCGC*T (SEQ ID N0:9)
gcgacccuGAuGaggccgugaggccGaaIgauuc*T (SEQ ID N0:19)
k2 (min') Substrate sequence
CCC < 0.01 Fl-GAAUCCCGGUCGC*T (SEQ ID NO:10)
CCA 0.04 Fl-GAAUCCAGGUCGC*T (SEQ ID NO:lI)
CCU <0.01 FI-GAAUCCUGGUCGC*T (SEQ ID N0:12)
gcgacccuGAuGaggccgugaggccGaaIaauuc*T (SEQ ID N0:20)
k2 (min') Substrate sequence
UCC <0.01 Fl-GAAUUCCGGUCGC*T (SEQ ID N0:13)
UCA < 0.01 FI-GAAUUCAGGUCGC*T (SEQ ID N0:14)
UCU < 0.01 Fl-GAAUUCUGGUCGC*T (SEQ ID NO: i5)
gcgacccuGAuGaggccgugaggccGaaAcauuc*T (SEQ ID N0:21)
k2 (mini'} Substrate sequence
GUC 0.13 Fl-GAAUGUCGGUCGC*T (SEQ ID N0:16)
Fl = Fluorescein label
*T = 3'-3' inverted thymidine
A, C, G, I, U = ribonucleotides (I is inosine)
a, c, g, a = 2'-O-allyl-ribonucleotides
The results show that substrates with A'6~zC'6~'H" and G'~ ZC'6~'H"
triplets are cleaved at a high rate. Comparison to the control catalytic
oligomer having an A at position 15.1 (to cleave a substrate with a
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G'6.zU16.'C" triplet) shows that substrates with A'6~ZC'6.'A" and G'6~zC'6.'A"
triplets (to be cleaved by a catalytic oligomer with an I at position 15.1)
have
an initial rate of cleavage that is higher than the corresponding control
reactions involving reactants with a standard A's.'-U16.1 base pair.
Example 2: Rate of cleavage at GCH and ACH triplets by catalytic
oligomers containing only ribonucleotides
Cleavage of substrate RNA by all-ribonucleotide versions of oligomers
(no modifications) designed to cleave GCH and ACH triplets was assessed.
The oligomers used were SEQ ID N0:17 (cleaves after ACH triplets) (Table
1) and SEQ ID N0:18 (cleaves after GCH triplets) (Table 1). The
corresponding short fluorescent labelled substrates, SEQ ID NOS:4, 5, and 6
(containing ACH triplets) (Table 1) and SEQ ID NOS:7, 8, and 9 (containing
GCH triplets) (Table 1) were used with catalytic oligomers SEQ ID N0:17
and SEQ ID N0:18 respectively.
The reactions were performed under single-turnover kinetic
conditions, using 2.5 ~,M catalytic oligomer and 250 nM substrate, both at
pH 6.0 in the presence of 10 mM Mg2+ and also at pH 7.4 in the presence of
1 mM Mg2+. All other reaction conditions were as in Example 1.
The data were analyzed as in Example 1. The k2 values for the 6
combinations of all-ribonucleotide catalytic oligomer and corresponding
substrates are shown in Table 4.
Table 4
kz (min)-'
Triplet pH 6.0; 10 mM Mgz+ pH 7.4; 1 mM Mg2+
GCA 0.39 2.32
GCC 0.18 2.03
GCU 0.03 0.10
ACA 0.41 1.12
ACC 0.17 0.71
ACU 0.02 0.06
The all ribonucleotide oligomers targeting GCA, GCC, ACA, and ACC sites
have a higher rate of cleavage than the all ribonucleotide oligomers targeting
GCU or ACU sites. Furthermore, Table 4 indicates the cleavage activity is
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very high in the presence of 1 mM Mg2+. This level of Mg2+ is similar to
the concentration of Mg2+ in vivo.
Example 3: Rate of cleavage at GCH and ACH triplets by catalytic
oligomers containing only 6 ribonucleotides
Cleavage of substrate RNA by 6-ribonucleotide versions of oIigomers
designed to cleave GCH and ACH triplets was assessed. In these oligomers,
ribonucleotides were present at positions U', G5, Ab, Ge, G'2 and I'S.' and
all
other positions were 2'-O-allyl-ribonucleotides (see Figure 2 for numbering).
The oligomers used were SEQ ID N0:17 (cleaves after ACH triplets) (Table
1 ) and SEQ ID N0:18 (cleaves after GCH triplets) (Table 1 ). The
corresponding short fluorescent labelled substrates, SEQ ID NOS:4, 5, and 6
(containing ACH triplets) (Table 1) and SEQ ID NOS:7, 8, and 9 (containing
GCH triplets) (Table 1 ) were used with catalytic oligomers SEQ ID N0:17
and SEQ ID N0:18 respectively.
I5 The reactions were performed under single-turnover kinetic
conditions, using 2.5 ~cM catalytic oligomer and 250 nM substrate, both at
pH 6.0 in the presence of 10 mM Mg2+ and also at pH 7.4 in the presence of
1 mM Mgz+. All other reaction conditions were as in Example 1.
The data were analyzed as in Example 1. The calculated k~ values for
the six combinations of 6-ribo catalytic oligomers and corresponding
substrates are shown in Table 5.
Table 5
kz (min)-'
Triplet pH 6.0; 10 mM Mg2+ pH 7.4; 1 mM Mg2+
GCA 0.11 0.87
GCC 0.03 0.14
GCU 0.03 0.11
ACA 0.18 1.06
ACC 0.01 0.12
ACU 0.02 0.08
These results indicate that catalytic oligomers such as SEQ ID N0:17
(cleaves after ACH triplets) (Table 1) and SEQ ID N0:18 (cleaves after
GCH triplets) (Table 1) remain active when all of the ribonucleotides except
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those at positions U4, G5, A6, G8, G'z and I'S~' are modified. These results .
also indicate that catalytic oligomers are capable of cleaving substrates at
physiological concentrations of Mg2+
Example 4: Rate of cleavage at a GCA triplet by versions of a catalytic
S oligomer differing in the sugar modification at position U4
Cleavage of substrate RNA by oligomers with modifications at
position U~ was assessed. These assays showed that oligomers that contain
nuclease resistant modifications retain activity and that different
modifications
can be made at a given position of the catalytic oligomer while retaining
activity. The catalytic oligomers were based on SEQ ID N0:18. Variant I
was made up of SEQ ID N0:18 with all but five nucleotides modified with
2'-O-allyl-ribonucleotides (ribonucleotides were present at positions G5, A~,
Ge, G'Z and I'S~' and all other positions were 2'-O-allyl-ribonucleotides; see
Figure 2 for numbering). Variant II was made up of SEQ ID N0:18,
modified with 2'-O-allyl-ribonucleotides at all but six nucleotides
(ribonucleotides were present at positions U4, G5, A6, Ge, G'Z and I'S'' and
all
other positions were 2'-O-allyl-ribonucleotides). Variant III contained
ribonucleotides at positions Gs, A6, G8, G'2 and I'S~' and a 2'-amino-2'-
deoxyuridine at position U4 . All other bases were 2'-O-allyl-ribonucleotides.
The reactions were performed under single-turnover kinetic
conditions, using 2.5 ~.M catalytic oligomer and 250 nM substrate, both at
pH 7.4 in the presence of 1 mM Mg2+. All other reaction conditions were
as in Example 1.
The data were analyzed as in Example 1. The calculated kz values
were 2.32 miri' for the all-ribonucleotide compound, 0.10 miri' for variant I,
0.87 min ' for variant II and 0.56 miw' for variant III respectively. These
results indicate that different modifications which inhibit RNase A activity
can be made at the highly RNase A sensitive U° site while retaining
activity.
The nuclease resistant variant containing the 2'-amino-2'-deoxyuridine at
position U4 is only marginally less active than the RNase A sensitive but
highly active variant which contains a ribouridine at this position. This
CA 02295207 1999-12-15
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demonstrates that it is possible to generate nuclease resistant catalytic
oligomers with activities close to the unmodified all-ribonucleotide versions,
and that different modifications can be made at a given site while retaining
activity.
Example 5: Comparison of cleavage activities of 2'-O-methyl- and 2'-O-
ailyl-modified catalytic oligomers at all 12 possible NCH
triplets
Cleavage of substrate RNA by oligomers with modifications in the
nucleotides at all positions except G5, A6, Gg, G'z, and I'S~' were assayed to
show cleavage at all NCH sites. Catalytic oligomers were based on SEQ ID
N0:17 (cleaves after ACH triplets), SEQ ID N0:18 (cleaves after GCH
triplets), SEQ ID N0:19 (cleaves after CCH triplets) and SEQ ID N0:20
(cleaves after UCH triplets) that either contained 2'-O-allyl-ribonucleotides
or 2'-O-methyl-ribonucleotides at all positions accept G5, A6, Gg, G'2, and
I'S~' were synthesized. The respective fluorescent labelled substrates SEQ ID
NOS:4, 5 and 6 (containing ACH triplets, cleaved by SEQ ID N0:17), SEQ
ID NOS:7, 8 and 9 (containing GCH triplets, cleaved by SEQ ID N0:18),
SEQ ID NOS:10, 11 and 12 {containing CCH triplets, cleaved by SEQ ID
N0:19) and SEQ ID NOS: 13, 14 and 15 (containing UCH triplets, cleaved
by SEQ ID N0:20) were also synthesized. The sequences of SEQ ID NOS:
4-20 are shown in Table 1.
The reactions were performed under single-turnover kinetic
conditions, using 2.5 ~,M catalytic oligomer and 250 nM substrate, at pH 7.4
in the presence of 10 mM magnesium ions. All other reaction conditions
were as in Example 1. The data were analyzed as in Example 1.
The results from these assays indicated that catalytic oligomers
directed at GCH, ACH, CCH, and UCH substrates are capable of cleaving
. their respective targets. The results also indicate that for many catalytic
oligomer:substrate combinations there is virtually no difference between the
2'-O-allyl-ribonucleotide modified catalytic oligomers and the corresponding
2'-O-methyl-ribonucleotide catalytic oligomers. Furthermore, for no
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combination is there much more than a 2-fold difference between these two .
types of modifications.
Example 6: Rate of cleavage at a GCA triplet by versions of a catalytic
oligomer differing in the nucleobase at position N'
Cleavage assays were performed to show that different bases with
different modifications could be substituted at position N' and still retain
activity. All catalytic oligomers contained 2'-O-allyl-ribonucleotides at all
positions except Gs, A6, Gg, G'Z and I's.' which were ribonucleotides. All
catalytic oligomers were designed to cleave after GCH triplets. The
sequences of the catalytic oligomers were as follows (the N' position is
marked in boldface):
L-gcgacccuGAuGaggccgugaggccGaaIcauuc*T(SEQ ID NO:18)
L-gcgacccuGAcGaggccgugaggccGaaIcauuc*T(SEQ ID N0:22)
L-gcgacccuGAaGaggccgugaggccGaaIcauuc*T(SEQ ID N0:23)
L-gcgacccuGAgGaggccgugaggccGaaIcauuc*T(SEQ ID N0:24)
L-gcgacccuGAiGaggccgugaggccGaaIcauuc*T(SEQ ID N0:25)
L-gcgacccuGAqGaggccgugaggccGaaIcauuc*T(SEQ ID N0:26)
L-gcgacccuGAnGaggccgugaggccGaaIcauuc*T(SEQ ID N0:27)
L = 5'-terminal hexanediol linker
*T = 3'-3' inverted thymidine
A, G and I are ribonucleotides
a, c, g, a and i are 2'-allyloxy-2'-deoxyribonucleotides
q is 1-(2-O-allyl-/3-D-ribofuranosyl)quinazoline-2,4-dione
n is 5-nitro-1-(2-O-allyl-/3-D-ribofuranosyl)indole
The reactions were performed under single-turnover kinetic
conditions, using 2.5 p,M catalytic oligomer and 250 nM substrate, at pH 7.4
in the presence of 10 mM magnesium ions. All other reaction conditions
were as in Example 1. The data were analyzed as in Example 1.
Graphs showing fraction product versus time curves of the N' variant
oligomers are shown in Figures 4A and 4B. The data indicate that all
variants at N' are capable of cleaving very well. The catalytic oligomers
tolerate bulky groups such as the uracil analogue, quinazoline-2,4-dione quite
well. Information such as this is important because it shows that there is a
position in the catalytic oligomers which can be varied to optimize catalytic
structure for a given substrate.
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Example 7: In vitro cleavage of a long substrate derived from hepatitis.
C virus
A long RNA substrate transcribed from a plasmid was used to
demonstrate cleavage of such a substrate using the disclosed compositions.
Plasmid pN(1-4728) contains the first 1358 bases of the hepatitis C virus
genome (HCV). The sequence is oriented so as to ailow a runoff transcript
of the Bam HI-linearized plasmid that produces a 1358 base transcript. A
runoff transcription (3-5 ~,g) was performed in a 100 ~,1 reaction volume
containing 40 mM Tris-HCl (pH 7.5), 18 mM MgCl2, 1 mM spermidine, 5
mM DTT, 2000 U/mI placental RNase inhibitor (Promega), 3 mM of each of
ATP, UTP, CTP and GTP, 50 ~.Ci of [a 32P]GTP (DuPont NEN) and 3000
U/ml T7 RNA poiymerase (New England Biolabs). The reaction was
incubated at 37°C for 2-3 hours and then terminated by addition of 100
~,l
RNA gel-loading buffer (98 % formamide, 10 mM EDTA, 0.025 % xylene
cyanol FF and 0.025 % bromophenol blue). The mixture was then heated at
90°C for three minutes, snap-cooled on ice and subjected to
electrophoresis
on a 4 % polyacrylamide/7 M urea gei. The 1358 nucleotide long transcript
was visualized by UV shadowing, the band was excised and the RNA
extracted by electroelution for one hour. The RNA was then recovered by
overnight ethanol precipitation. After centrifugation and washing with 70 %
ethanol the purified transcript was resuspended in 20 tcl of DEPC sterilized
water and the concentration was determined by UV measurement.
The following oligomers were designed to target the runoff transcript
of the HCV.
L-ggauucgcuGAuGaggccgugaggccGaaIcucaugg*T (SEQ ID N0:28)
L-gauucgcuGAaGaggccgugaggccGaaIcucaugg*T (SEQ ID N0:29)
L-ggauu~gcUGAuGaggccgugaggccGaaIcucaugg*T (SEQ ID N0:30)
L-gauucgcuGAuGaggccgugaggccGaaIcucaugg*T (SEQ ID N0:3I)
L = 5'-terminal hexanediol linker
*T = 3'-3' inverted thymidine
A, G, U and I are ribonucleotides
a, c, g and a are 2'-allyloxy-2'-deoxyribonucleotides
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These sequences are targeted to the GCA-345 site of the HCV genome
which is present in the transcript.
Cleavage reactions were carried out in a 10 p.l volume containing 50
mM Tris-HCI (pH 7.5), 10 mM MgClz, 30 nM radiolabeled transcript and
S 300 nM catalytic oligomer (either SEQ ID NOS:28-31). Incubation was
performed at 37°C for 10 minutes and 60 minutes for each oligomer
tested,
and the reactions were quenched by addition of 10 ~.1 of gel-loading buffer
containing 20 mM EDTA, heated at 90°C for 5 minutes and then cooled on
ice. Uncleaved transcript and cleavage products were then separated by
electrophoresis on a 4 % polyacrylamide/7 M urea gel. After electrophoresis
the gel was transferred onto Whatman 3MM filter paper and dried for two
hours at 80°C. Bands were quantitated by exposure to a PhosphorImager
screen.
The results of these assays indicated that all four of the catalytic
oligomers were capable of cleaving the HCV runoff transcript. This
indicates that target catalytic oligomers containing all 2'-O-allyl-
ribonucleotides, except at positions U°, G5, A6, Gg, G'2 and I'SV (SEQ
ID
N0:30) and except at positions G5, A6, Ge, G'2 and I'S~' (SEQ ID NOS:28,
29, and 31), are capable of cleaving a long RNA.
Example 8: Comparison of in vitro cleavage of a long substrate derived
from hepatitis C virus using catalytic oligomers targeting
GCA and GUA triplets
Cleavage assays were performed to show that catalytic oligomers
designed to cleave the 1358 base HCV substrate function at concentrations as
low as 30 nM. The preparation of the runoff transcript was as in Example 7.
The oligomers used in these assays were as follows:
35
L-ggauucgcuGAuGaggccgugaggccGaaIcucaugg*T (SEQ ID N0:28)
L-uuggugucuGAuGaggccgugaggccGaaAcguuugg*T (SEQ ID N0:32)
L = 5'-terminal hexanediol linker
*T = 3'-3' inverted thymidine
A, G, U and I are ribonucleotides
a, c, g and a are 2'-allyloxy-2'-deoxyribonucleotides
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These catalytic oligomers target the GCA 345 triplet (oligomer of
SEQ ID N0:28) and the GUA 378 triplet (oligomer of SEQ ID N0:32). The
fragments obtained when analyzing cleavage reactions of the 1358 runoff
transcript of the HCV are 347 and 1011 nucleotides long for cleavage at the
GCA 345 site and 380 and 978 nucleotides long for cleavage at the GUA 378
site. The catalytic oligomers were compared at 1 and 3 ~,M with a reaction
time of one hour and at concentrations of 30 nM, 100 nM, 300 nM, 1 ~,M
and 3 ~,M with a reaction time of three hours. All other reaction and
conditions were as in Example 7.
An analysis of the data indicated that the oligomers cleaved the HCV
transcript after three hours at all concentrations of catalytic oligomer
tested.
This indicates that concentrations of catalytic oligomer of 30 nM are capable
of cleaving a 1358 base RNA fragment of the HCV genome.
Example 9: Cleavage of human IL-2 mRNA in Jurkat cell lysates
Cleavage assays were performed to show that catalytic oligomers
targeted to IL-2 mRNA were capable of cleaving the native mRNA in a cell
lysate solution. These assays were performed with the catalytic oligomer of
sequence:
L-gacuuagcuGAuGaggccgugaggccGaaIcaaugca*T (SEQ ID N0:33)
L = 5'-terminal hexanediol linker
*T = 3'-3' inverted thymidine
A, G, U and I are ribonucleotides
a, c, g and a are 2'-allyloxy-2'-deoxyribonucleotides.
This sequence was designed to cleave after the GCA, where the A is at
position 140, in the human interleukin-2 mRNA (sequence name HSIL2R in
the EMBL Nucleotide Sequence Database 43'd Edition).
Jurkat cells were stimulated for five hours with phorbol 12-myristate
13-acetate (PMA)/phytohemoagglutinin (PHA) to induce expression of IL-2.
Crude cell lysates were then prepared by freeze-thawing as follows: 2 X 10'
cells were washed in phosphate buffered saline (PBS) and resuspended in 500
ul of RNase free deionized water, incubated at room temperature for 10
minutes, then snap frozen in liquid nitrogen and thawed at 37°C. Cell
debris
CA 02295207 1999-12-15
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was removed by low speed centrifugation leaving a crude lysate for use in .
cleavage assays.
Each cleavage reaction was carried out at 37°C in a 100 ,ul
reaction
volume including 62.5 ~l of cell lysate, 50 mM Tris pH 7.5, 10 mM MgClz
and 1 ~,M catalytic oligomer for the reaction times: 0, 10, 30, 60, and 120
minutes. The following controls were done, control after 120 minutes with
no oligomer, IL-2 probe control, IL-2 and ~3-actin RNase digested, and a
control after 10 minutes with no oligomer. RNA was purified from the
reactions using BioGene X-Cell solution and analyzed by a ribonuclease
protection assay (RPA), using the RPA II kit from Ambion, following the
manufacturer's protocol. Biotin labelled antisense RNA probes for IL-2 and
a-actin RNA (internal standard) were prepared using an SP6 transcription kit.
The template for the (3-actin probe was purchased from Ambion and produced
an RNA probe of 334 nucleotides and a protected fragment of 245
nucleotides in length.
An IL-2 probe template was made by using RT-PCR to amplify a
fragment of the IL-2 sequence from Jurkat cell RNA. One primer was
designed to also include the SP6 transcription promoter site so that the
resultant DNA probe could be transcribed directly from the PCR reaction.
The probe was designed to be 487 nucleotides in length, leading to a
protected fragment also of 487 nucleotides after RPA analysis of IL-2 RNA.
RPA analysis after cleavage with the catalytic oligomer should identify
protected fragments of 428 and 59 nucleotides in addition to the full length
RNA.
Protected RNA fragments were separated by polyacrylamide gel
electrophoresis (5 % ), blotted onto nylon membrane and visualized by
chemiluminescent detection using the BrightStar Biodetect kit from Ambion.
Biotinylated RNA markers of lengths 500, 400, 300 and 200 nucleotides were
used. The cell lysate prepared as described above contains, the IL-2 mRNA
produced through intracellular transcription. This cell lysate, contains the
targeted substrate. The product of the reaction between. the substrate and the
catalytic oligomer produces a 428 base and 59 base fragment of the IL-2
41
CA 02295207 1999-12-15
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mRNA. By using a labeled probe for this sequence, the cleavage products .
can be detected. The IL-2 mRNA was cleaved by the catalytic oligomer,
SEQ ID N0:33, in the presence of the cell lysate. This indicates that the
catalytic oligomers are capable of cleaving a long, native, mRNA in the
presence of the cellular material associated with the mRNA in vivo.
Example 10: Cleavage of rat dopamine D2 receptor RNA in CHO cell
lysates
Cleavage assays were performed to show that catalytic oligomers
targeted to rat dopamine D2 receptor mRNA were capable of cleaving the
native mRNA in a CHO cell lysate solution. The assays were performed
with the catalytic oligomer of sequence:
L- gcucgaccuGAuGaggccgugaggccGaaIcugcgcu*T (SEQ ID N0:34)
L = 5'-terminal hexanediol linker
*T = 3'-3' inverted thymidine
A, G, U and I are ribonucleotides
a, c, g and a are 2'-allyloxy-2'-deoxyribonucleotides.
This sequence was designed to cleave after the GCA, where the A is at
position 811, in the rat dopamine D2 receptor RNA (sequence name
RND2DOPR in the EMBL Nucleotide Sequence Database 43'° Edition).
Crude cell lysates were made from CHO cells stably transfected with
the rat dopamine D2 receptor gene by the freeze-thawing procedure as
described in Example 9 above. Each cleavage reaction was carried out at
37°C in a 100 ~,1 reaction volume including 62.5 ~.l of cell lysate, 50
mM
Tris pH 7.5, 10 mM MgCl2 and 0.5 p.M catalytic oligomer for the reaction
times: 10, 30, 60, and 120 minutes The following controls were performed:
dopamine D2 receptor RNA and ~i-actin digested with RNase, dopamine D2
receptor RNA and /3-actin control, rat dopamine D2 receptor RNA probe
and a-actin control, control after 10 minutes with no oligomer, control after
30 minutes with no oligomer, and control after 60 minutes with no oligomer.
RNA was purified from the reactions and analyzed by ribonuclease
protection assay, using the RPA II kit from Ambion and following the
manufacturer's protocol. Biotin labelled antisense RNA probes for rat
42
CA 02295207 1999-12-15
WO 98/58058 PCT/US98112663
dopamine D2 receptor and mouse ~i-actin RNA (internal standard) were
prepared using an SP6 transcription kit. The template for the ~i-actin probe
was purchased from Ambion and produced an RNA probe of 334 nucleotides
and a protected fragment of 245 nucleotides in length.
A dopamine D2 receptor probe template was made by using RT-PCR
to amplify a fragment of the dopamine D2 receptor sequence from CHO cell
RNA. One primer was designed to also include the SP6 transcription
promoter site so that the resultant DNA probe could be transcribed directly
from the PCR reaction. The probe was designed to be 663 nucleotides after
RPA analysis of dopamine D2 receptor RNA. RPA analysis after cleavage
with the catalytic oligomer should identify protected fragments of 394 and
269 nucleotides in addition to the full length RNA. Protected RNA
fragments were separated by polyacrylamide gel electrophoresis (5 %), blotted
onto nylon membrane and visualized by chemiluminescent detection using the
BrightStar Biodetect kit from Ambion.
The results indicated that the D2 receptor RNA is cleaved by the
catalytic oligomer, SEQ ID N0:34. The cleavage products were detectable
by 10 minutes and increased with time indicating that the catalytic oligomer
is not being substantially degraded over time.
Example 11: Cleavage of human ICAM-1 mRNA in A549 cell lysates
Cleavage assays were performed to show that catalytic oligomers
targeted to ICAM-1 mRNA were capable of cleaving the native mRNA in an
A549 cell lysate solution. The assays were performed with the catalytic
oligomers of sequences:
L-ugguucucuGAuGaggccgugaggccGaaIuguauaa*T (SEQ ID N0:35)
L-uguaguccuGAuGaggccgugaggccGaaIuauuucu*T (SEQ ID N0:36)
L = 5'-terminal hexanediol linker
*T = 3'-3' inverted thymidine
A, G, U and I are ribonucleotides
a, c, g and a are 2'-allyloxy-2'-deoxyribonucleotides.
These sequences were designed to cleave after the ACA sites where
the first A is positioned at base 1205 (SEQ ID N0:35) and position 1592
43
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WO 98/58058 PCT/US98/12663
(SEQ ID N0:36), in the human intercellular adhesion molecule-1 mRNA
(ICAM-1) (sequence name HSICAMO1 in the EMBL Nucleotide Sequence
Database 43'd Edition).
Crude cell lysates were made from A549 cells, after a five hour
stimulation with 10 ng/ml of hTNF« to induce expression of ICAM-1, by the
freeze-thawing procedure as described in Example 9 above. Each cleavage
reaction was carried out for two hours at 37°C in a 100 ~,1 reaction
volume
including 62.5 pl of cell lysate, 50 mM Tris pH 7.5, 70 mM MgCI2 and
catalytic oligomer to a final concentration of either 500 nM oligomer, 200
nM oligomer, 100 nM oligomer, 50 nM oIigomer, control without catalytic
oligomer. RNA was purified from the reactions and analyzed by
ribonuclease protection assay, using the RPA II kit from Ambion, following
the manufacturer's protocol. Biotin labelled antisense RNA probes for
human ICAM-1 and GAPDH RNA (internal standard) were prepared using
an SP6 transcription kit. The template for the GAPDH probe was purchased
from Ambion and produced a protected fragment of 316 nucleotides in
length.
An ICAM-1 probe template was made by using RT-PCR to amplify a
fragment of the ICAM-1 sequence from A549 cell RNA. One primer was
designed to also include the SP6 transcription promoter site so that the
resultant DNA probe could be transcribed directly from the PCR reaction.
The probe was designed to be 598 nucleotides in length, leading to a
protected fragment also of 598 nucleotides after RPA analysis of ICAM-1
RNA. RPA analysis after cleavage with the catalytic oligomers should
identify protected fragments of 552 and 46 nucleotides (1205 ACA site) and
433 and 165 nucleotides ( I592 ACA site) respectively in addition to the full
length RNA.
Protected RNA fragments were separated by 5 % polyacrylamide gel
electrophoresis, blotted onto nylon membrane and visualized by
chemiluminescent detection using the BrightStar Biodetect kit from Ambion.
Biotinylated RNA markers of length 500, 400, 300 and 200 nucleotides were
used. Cleavage products were produced at all concentrations of oligomer
44
CA 02295207 1999-12-15
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tested. Cleavage at the 1592 ACA site was particularly effective even using
only 50 nM catalytic oligomer.
CA 02295207 1999-12-15
WO 98/58058 PCT/US98/-12663
SEQUENCE LISTING
(1)
GENERAL
INFORMATION:
(i) APPLICANT: Innovir Laboratories, Inc.
(ii) TITLE OF INVENTION: Compositions Having Activity
RNA-Cleavage
(iii) NUMBER OF SEQUENCES: 36
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Patrea L. Pabst
(B) STREET: 2800 One Atlantic Center
1201 West Peachtree Street
(C) CITY: Atlanta
(D) STATE: GA
(E) COUNTRY: USA
(F) ZIP: 30309-3450
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy dis3c
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version
#1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Pabst, Patrea L.
(B) REGISTRATION NUMBER: 31,284
(C) REFERENCE/DOCKET NUMBER: ILI 123
(ix} TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (404)-873-8794
(B) TELEFAX: (404)-873-8795
(2)
INFORMATION
FOR
SEQ
ID
NO:
1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: circular
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
rf111Nr~NNNNNN NNNNNNCUGA NGPSJRNNNNN rf117VNNNNyNG 57
p~~I~NNNNNNN NNNNNUH
(2)
INFORMATION
FOR
SEQ
ID
NO:
2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
NNNNNNCUGA 35
NGF~JRNNNNN
NNNNNYNGAA
NNNNN
(2)
INFORMATION
FOR
SEQ
ID
NO:
3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B)- TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
NNNNCHNNNN 12
NN
(2)
INFORMATION
FOR
SEQ
ID
NO:
4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
46
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WO 98/58058 PCT/US98/12663
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: .
GAAUACCGGU CGCT 14
(2) INFORMATION FOR SEQ ID NO: 5:
, (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
GAAUACAGGU CGCT 14
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GAAUACUGGU CGCT 14
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GAAUGCCGGU CGCT 14
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GAAUGCAGGU CGCT 14
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GAAUGCUGGU CGCT 14
(2) INFORMATION FOR SEQ ID N0: 1~:
(i) SEQUENCE CHARACTERISTICS:
. (A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
. (C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GAAUCCCGGU CGCT 14
(2) INFORMATION FOR SEQ ID N0: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
47
CA 02295207 1999-12-15
WO 98/58058 PCT/US98/-12663
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID NO: 11:
SEQ
GAAUCCAGGU CGCT
14
(2) INFORMATION FOR SEQ ID
NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi} SEQUENCE DESCRIPTION: ID NO: 12:
SEQ
GAAUCCUGGU CGCT 14
(2) INFORMATION FOR SEQ ID
NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID NO: 13:
SEQ
GAAUUCCGGU CGCT 14
(2} INFORMATION FOR SEQ ID
NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID NO: 14:
SEQ
GAAUUCAGGU CGCT 14
(2) INFORMATION FOR SEQ ID
NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID NO: 15:
SEQ
GAAUUCUGGU CGCT 14
(2} INFORMATION FOR SEQ ID
NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID NO: 16:
SEQ
GAAUGUCGGU CGCT 14
(2) INFORMATION FOR SEQ ID
NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: ID NO: 17:
SEQ
GCGACCCUGA UGAGGCCGUG AGGCCGAANU 35
AUUCT
48
CA 02295207 1999-12-15
WO 98/58058 PCT/US98/12663
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ NO: 18:
ID
GCGACCCUGA UGAGGCCGUG AGGCCGAANC 35
AUUCT
(2) INFORMATION FOR SEQ ID NO:
19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ NO: 19:
ID
GCGACCCUGA UGAGGCCGUG AGGCCGAANG 35
AUUCT
(2) INFORMATION FOR SEQ ID NO:
20:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ NO: 20:
ID
GCGACCCUGA UGAGGCCGUG AGGCCGAANA 35
AUUCT
(2) INFORMATION FOR SEQ ID NO:
21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ NO: 21:
ID
GCGACCCUGA UGAGGCCGUG AGGCCGAAAC 35
AUUCT
(2) INFORMATION FOR SEQ ID NO:
22:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ N0:22
ID
GCGACCCUGA CGAGGCCGUG AGGCCGAANC 35
AUUCT
(2) INFORMATION FOR SEQ ID NO:
23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ N0:23
ID
GCGACCCUGA AGAGGCCGUG AGGCCGAANC 35
AUUCT
(2) INFORMATION FOR SEQ ID NO:
24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
49
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WO 98/58058 PCT/US98/12663
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24
GCGACCCUGA GGAGGCCGUG AGGCCGAANC AUUCT 35
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25
GCGACCCUGA NGAGGCCGUG AGGCCGAANC AWCT 35
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26
GCGACCCUGA NGAGGCCGUG AGGCCGAANC AUUCT 35
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27
GCGACCCUGA NGAGGCCGUG AGGCCGAANC AUUCT 35
(2) INFORMATION FOR SEQ ID NO: 28:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28
GGAUUCGCUG AUGAGGCCGU GAGGCCGAAN CUCAUGGT 3g
(2) INFORMATION FOR SEQ ID NO: 29:
(i} SEQUENCE CHARACTERISTICS:
(A} LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29
GAUUCGCUGA AGAGGCCGUG AGGCCGAANC UCAUGGT 37
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30
GGAUUCGCUG AUGAGGCCGU GAGGCCGAAN CUCAUGGT 3g
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
CA 02295207 1999-12-15
WO 98/58058 PCT/US98/12663
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31
GAUUCGCUGA UGAGGCCGUG AGGCCGAANC UCAUGGT 37
(2) INFORMATION FOR SEQ ID NO: 32:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32
UUGGUGUCUG AUGAGGCCGU GAGGCCGAAA CGUUUGGT 38
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33
GACUUAGCUG AUGAGGCCGU GAGGCCGAAN CAAUGCAT 38
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs ,
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:34
GCUCGACCUG AUGAGGCCGU GAGGCCGAAN CUGCGCUT 38
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35
UGGUUCUCUG AUGAGGCCGU GAGGCCGAAN UGUAUAAT 38
(2) INFORMATION FOR SEQ ID NO: 36:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQU$NCE DESCRIPTION: SEQ ID N0:36
UGUAGUCCUG AUGAGGCCGU GAGGCCGAAN UAUUUCUT 38
$1
