Note: Descriptions are shown in the official language in which they were submitted.
'.'~,`',;;,Ct~.:. ~1~9626
~"'~.WO94~15~ '^ PCT/US93~1117B
DESCRIPTION
''~
Formation~Of Triple Helix Com~lrexes of
Sinqle Stranded Nucleic Acids Usinq Nucleoside
,O~liqo~ls~ W-lcs~5S~D~_se Pyrimidine Analoqs
,
Cross=Reference to Related Applications
This application is a continuation-in-part of U.S.
Serial No. 07/772,081, filed October 7, l99l, which is a
continuation-ln-part of U.S. Serial No. 368,027, filed
June l9, 1989, which is a`continuation-in-part of U.S.
Serial No. 924,234, filed October 28, 1986, the disclo-
~- sures of which are incorporated herein by reference.
Back~round_and Introduction to the Invention
This invention was made with federal governmental
support, including grants~from~the Department of Ener~y
and the NIH/National Cancer Institute, Grant Numhers DE-
FG02-B8ER60636 and 2P0lCA42~762-04AI. The Government has
certain rights to this application.
Publications and other re,~erence materials referred
to herein are~in~orporated~herein by r:eference.
' The present~invention is directed'to novel methods of
detecting,~ recognizing and/or inhibiting or altering
expression of spec-ific sequences in single stranded
nucleic acids, particularly ~NA, using Second and Third
2Q Strands which are capable of specifically complexing with
a selected slngle stranded nucleic acid sequence to give
a triple helix complex.
Formation of triple helix complexes by homopyrimidine
oligodeoxynucleotides binding to polypurine tracts in
~ouble-stranded DNA'by Hoogsteen hydrogen bonding has been
~ ` :
'~ reported. See, e.q., Moser, H.E., et al., Science
, 233-645-650 (1~87) and Povsic, T.J., et al., J. Am. Chem.
Soc. lll:3059-306l' (1989~. The homopyrimidine oligo
' nucleotides were said to recognize extended purine
sequences in the major groove of double helical DNA via
v ~ i
WO94/11534 Z~9 6 2 ~ PCT/US93/l1178
triple helix formation. Specificity was said to be
imparted by Hoogsteen base pairing between the homopyri-
midine oligonucleotide and the purine strand of the
Watson-Crick duplex. DNA triple helical complexes con-
taining cytosine and thymidine on the third strand havebeen reported to be stabl~ in slightly acidic to neutral
solutions (pH 5.0-6.5), respectively, but have been
reported to dissociate on increasing pH. Incorporation of
modified bases of T, such as 5-bromo-uracil, and C, such
as 5-methylcytosine, into the third strand has been
- reported to increase stability of the triple helix over a
higher pH range: In order for cytosine (C) to participate
in the Hoogsteen-type pairing, it was thought that a
hydrogen must be available on the N-3 of the pyrimidine
ring for hydrogen bonding. Accordingly, it has been
proposed that cytosine be protonated at N-3.
DNA has been reported to exhibit a variety of
polymorphic conformations; such conformations may be
essential for biological processes. Modulation of signal
transduction by sequence-specific protein-DNA binding and
molecular interactions such as transcription, translation
and replication, are believed to be dependent upon DNA
conformation. ~eIls, R.D., et al., FASEB J. 2:2939-2949
(1988).
The possibillty of developing therapeutic agents
which bind to critical regions of the genome and
selectively i.nhibit the function, replication and survival
of abnormal cells is an exciting concept. See, e.a.,
Dervan, P., Science 232:464-471 ~1988).. Various
laboratories have pursued the design and development of
molecules which interact with DNA in a sequence-specific
manner. Such molecules have been proposed to have far-
~;` reaching implications for the diagnosis and treatment of
diseases involving foreign genetic materials (such as
viruses) or alterations to genomic DNA (such as cancer).
Nuclease-resistant nonionic oligodeoxynucleotides
~ODN) having a methylphosphonate backbone have been
21~9626
..{., ~
.WO94/11534 PCT/US93/11178
studied ln vitro and ln vivo as potential anticancer,
antiviral and antibacterial agents. Miller, P.S., et al.
Anti-Cancer ~rug Design, 2:117-128 (1987~. The 5'-3'
linked internucleoside bonds of these analogs are said to
approximate the conformation of phosphodiester bonds in
nucleic acids. With methylphosphonates, it has been
proposed that the phosphate backbone is rendered neutral
; by methyl substitution of one anionic phosphoryl oxygen,
which is thought to decrease inter and intra-strand
repulsion due to the charged phosphate groups. Miller,
P.S. et al.l Anti-Cancer Drug Design 2:117-128 (1987).
Oligodeoxynucleoside analogs with a MP backbone are
believed to penetrate living cells and have been reported
to inhibit mRNA transIation in globin synthesis and
vesicular stomatitis viral protein synthesis and to
inhibi~ splicing of pre-mRNA in inhibition of herpes
simplex virus (HSV) replication. Mechanisms of action for
inhibition by the MP analogs include formation of stable
complexes with complementary RNA and/or DNA.
Nonionic oligonucleoside alky~- and aryl-phosphonate
analogs complementary to a selected single stranded
foreign nucleic acid sequence are reported to be able to
selectîvely inhibit the function or expression of that
particular nucleic acid without disturbing the function or
expression of other nucleic acids present in the cell, by
binding to or interfering with that nucleic acid. (See,
e.q., U.S. Patent Nos. 4,469,863 and 4,511,713). The use
of complementar~ nuclease-resistant nonionic oligonucleo-
side methylphosphonates which are taken up by mammalian
cells to inhibit viral protein synthesis in certain
contexts, including Herpes simplex virus-l is described in
U.S. Patent No. 41757rO55.
The use of anti-sense oligonucleotides or phosphoro-
thioate analogs complementary to a part of viral mRNA to
interrupt the transcription and translation of viral mRNA
into~protein has been proposed. The anti-sense constructs
can bind to viral mRNA and were thought to obstruct the
. .
`
W094/lts34 21496~ PCT/IS93/11178 ~ I
cell's rihosomes from moving along the mRNA and thereby
halting the translation of mRNA into protein, a process
called 1'translation arrest'1 or '1ribosomal-hybridization
arrest. Il Yarochan, et l., "AIDS Therapies", Scientific
American, pages llO-ll9 ~October, l988).
The inhibition of infection of cells by HTLV-III by
administration of oligonucleotides complementary to highly
conserved regions of the HTLV-III genome necessary for
HTLV-III replication and/or expression is reported in U.S.
lO Patent No. 4,806,463. The oligonucleotides were said to
affect viral replication and/or gene expression as assayed
by reverse transcriptase activity (replication) and
production of viral proteins pl5 and p24 (gene
expression).
lS The ability of some antisense oligodeoxynucleotides
containing internucleoside methylphosphonate linkag2s to
inhibit HIV-induced syncytium formation and expression has
been studied. Sarin, et al., Proc. Nat. Acad. Sci. (USA)
85:744~-7451 (~988).
PCT Published Application W~ 9l/06626 described
oligonucleotides which are said to have tandem sequences
of inverted polarity and which are said to be useful for
forming an extended triple helix wi~h a double helical
nucleotide duplex. The inverted polarity was said to
stabilize the single strand oligonucleotides to exo-
nuclease degradation.
. .
Summary of the Invention
The present invention is directed to methods of
selectively detecting, recognizing and/or inhibiting or
altering expression of a specific target sequence of a
single ~tranded nucleic acid having any selected sequence
of nucleosides (e.g., mixed purine and pyrimidine nucleo-
sides) by formation of a triple helix complex wherein a
Second Strand specifically binds to the target sequence by
~;~ 35 Watson Crick base pairing and also specifically hydrogen
bonds to a Third Strand. The Second and Third Strands
.~
~;` 214~62~ -
t;:~3`~94~tS34 PCT/US~3/11178
comprise Oligomers that are optionally covalently linked
to each other.
Among other factors, the present invention is based
upon our finding that triple helix complexes may be formed
with a target sequence having any selected combination of
pyrimldine and purine bases using the hydrogen bonding
motifs described herein. In particular, according to one
aspect, nucleosides are used in the Second Strand which
have two hydrogen bonding~ faces, a Watson-Crick binding
face and a Second-Third Strand binding face. The Watson-
Crick binding.face of a Second Strand base hydrogen bonds
to the corresponding base of a nucleoside of the target
~equence by Watson-Crick base pairing. The Second-Third
Stand binding face of a Second Strand base has at least
two hydrogen bonding (donor and/or acceptor) sites and
; ~ specifically hydrogen bonds with and binds to a comple-
mentary base of a corresponding nucleoside of the Third
Strand. By use of this feature of the two hydrogen
bonding faces of the bases of the Second Strand, triplets
are formed and formation of mult~ple triplets gives a
triple helix complex. The naturally occurring pyrimidine
bases do not have sufficient hydrogen bonding sites on
what would be their Second-Third Strand binding faces to
stably hydrogen bond with a base of a Third Strand to form
a triplet. Since the purine bases, A and G have suffi-
cient hydrogen bonding sites on what would be their
Second-Third Strand binding faces, these "open sandwich"
type of triple helix complexes were thought to be
restricted to pyrimidine- rich target sequences in the
absence of other third strand modifications. According to
our proposed binding motifs, pyrimidine analogs are
provided which have an additional hydrogen bonding
(donor/acceptor) site on their Second-Third Strand binding
faces ("pyrimidine 5-donor/acceptor bases"). Thus, we
have found that by using nucleosldes having bases which
;~ are analogs of the naturally occurring cytidine and
~ uridine (or thymidine) in the Second Strand in place of
,~
2l4962~
WO94J11534 PCT/US93/111i8
those pyrimidines and which, unlike these naturally
occurring pyrimidines, have a proton donor or acceptor at
the 5-position; both a base of a nucleoside of the target
sequence and a base o~ a nucleoside of the Third Strand
can hydrogen bond to the Second Strand base and form a
triplet. The base of the corresponding target sequence
;nucleoside hydrogen bonds with the Watson-Crick binding
face of the Second Strand base and the base of the
corresponding Third Strand nucIeoside hydrogen bonds with
the Second-Third Strand binding face of the Second Strand
base. Use of these pyrimidine analogs advantageously
allows ~ormation of triple helix complexes with single
stranded nucleic acids having any target sequence (any
mixture of pyrimidine and purine nucleosides) without
~-~ 15 restriction of the target sequence to homopyrimidine or
`~ homopurine sequences or sequences having polypyrimidine or
polypurine tracts linked together. Thus, use of nucleo-
sides having pyrimidine 5-donor/acceptor bases in the
Second Strand and a`Third Strand selected according to one
of motifs I to V' of Figure 1 or ~igure 2B allows forma-
.
tion of a triple helix complex with a target sequence
which contains any mixture o~ pyrimidine and purine bases
.
In contrast, previously proposed protocols for triple
helix formation required either a target se~uence having
only purine bases or only pyrimidine bases, or if the
,
target sequence was comprised of a mixture of purine and
pyrimidine bases, it was thought necessary to use a Third
Strand having either lengthening links so as to be able to
switch from binding one~str~nd to the other strand or
having multiple reverses in strand polarity ~e.g.,
reversing from 5'-3' to 3'-5' and so forth~.
According to one aspect, the present invention is
directed to a method of detecting, recognizing or inhibit~
ing or altering expression of a specific target sequence
;~35 of single stranded nucleic acid having nucleosides
comprising both purine and pyrimidine bases. The single
stranded nucleic acid is contacted with Second and Third
21~962~ ~
WO94/11~34 PCT/US93/1137g
Strands which comprise Oligomers optionally linked
together. The Second Strand comprises at least one
nucleoside with a pyrimidine 5 donor/acceptor base. The
Second Strand is sufficiently complementary to the target
sequence and the Third Strand is sufficiently complemen-
tary to the Second Strand to form a triple helix complex
by formation of triplets between individual bases of the
target sequence and individual bases of each of the Second
and Third Strands.
According to a preferred aspect, the nucleoside
sequences of the Second and Third Strands are selected
according to one of motifs I to V' of Figure 1 or Figures
2A and 2B such that triplets are formed, each triplet
comprising a base of a nucleoside of the target sequence
hydrogen bonding with a base of a nucleoside of the Second
Strand and the Second Strand base hydroyen bonding with
both the target strand base and a base of a nucleoside of
the Third Strand. Formation of multiple adjacent triplets
produces a triple helix complex.
Thus, according to this aspec~, the present invention
is based upon our inno~ati~e finding that by selecting the
base sequences of the Second Strand and the Third Strand
according to one of the motifs I to V' of Figure 1 or
Figures 2A and 2B one may specifically detect or recognize
a target se~uence and form a triple helix complex with the
target sequence without regard to it~ base composition.
The Second and Third Strands having base sequences
3elected in accordance with one of motifs I to V' of
Figure l or Figure 2A and 2B exhibit high specificity and
high affinity in recognizing the target sequence and
formation of a triple helix complex at physiological pH
and temperatures.
In one aspect, the present invention provides a
Second Strand complementary to the target sequence which
binds to the target sequence by Watson-Crick base pairing
and which has in place of the naturally occurring pyrimi-
dine bases C and U (or T), modified pyrimidine bases which
2149625 ~
W~94/11~34 PCT/US93/11178
have an additional hydrogen bonding site at the 5-position
t"pyrimidine-5-donor/accePtor-bases"). (See Figure 3A for
the base numbering convention used herein). These
pyrimidine-5-donor/acceptor bases ha~e a structure which
allows them to act as a proton donor or acceptor at the 5-
position of the basels ring and which gives the base an
additional position for hydrogen bonding. These pyri-
midine-5-donor/acceptor bases have an additional hydrogen
bonding site and therefore can form hydrogen bonds and, ~-
thus, bind to another base (on its Second-Third Strand
binding face) according to one of motifs I to V' of Figure
l o~ Figure 2B. These pyrimidine 5-donor/acceptor bases
can bind to a;base of the target sequence by Watson Crick
base pairing on their front side (or "Watson-Crick binding
~ace") and also form hydrogen bonds on their back side (or
"Second-Third Strand binding face") with another base
according to one o~ motifs I to V' of Figure l or Figure
2B ~see "Third Strand Base Selection") to ~orm a triplet.
Thus, another aspect of the present invention is directed
to a Second Strand having at le~t one pyrimidine-5-
donor/acceptor base. Suitable pyrimidine-5-donor/acceptor
bases inclu~e pseudoisocytosine or pseudoisocytosine~ (in
place of cytosine) and pseudouracil or pseudocytosine (in
place of uracil or thymine). See Figure 4 for structures.
According to one embodiment, the Second Strand and
the Third 5trand may be covalently linked and, thus,
comprise a single Oligomer. According to an alternate
em~odiment, the~ Second Strand and the Third Strand may
each comprise separate Oligomers.
According to a preferred aspect, the Second and Third
Strands comprise substantially neutral Oligomers.
Especially preferred substantially neutral Oligomers are
methylphosphonate Oligomers. ~ ;
Preferably the Second and Third Strands each comprise
from about 4 t~ about 40 nucleosides, more preferably from
about 6 to about 30 nucleosides. Especially preferred are
,;
. 2149626
~i . wo ~4/tl53q PCT/US~3/11178
Second and Third Strands which each comprise a~out 8 to
I about 20 nucleosides.
! According to one aspect, the present invention is
directed to a Second Strand which is a first Oligomer and
which has a nucleoside sequence selected in accordance
with one of motifs I to V' of Figure l or Figure 2A.
According to an alternate aspect of the present
in~ention, a Second Strand capable of forming a triple
hellx complex with a target sequence having a mixture of
`lO purine and pyrimidine nucleosides is provided. The Second
Strand comprises a plurality of nucleosides wherein the
base portion of each nucleoside has a Watson-Crick binding
face capable of binding to a base of a nucleoside of the
target sequence by Watson-Crick base pairing and a Second-
Third Strand binding face having at least two hydrogen
binding sites and being capable of binding to a base of a
, nucleoside of the Third Strand.
~: .
t~ ~ According to another aspect, the present invention is
directed to a Third Strand which is a second Oligomer
ha~ing a nucleoside sequence selec~ed in accordance with
one of motifs~I to V' of Figure 1 or Fisure 2B.
Taken~together a base of a nucleoside of each of the
Second and Third Strands will interact with each other and
~ the base of the Second Strand will interact with a corres-
1~ 25 ponding base of a nucleoside of the target sequence to
i form a triplet as set forth in one of motif~ I to V' of
Figure 1 or Figure 2A and B. Formation of triplets with
~l ~ bases of multiple- adjacent nucleosides of the target
sequence result in a triple helix complex.
According to an alternate aspect of the present
invention, methods are provided of forming a triplet
~; between a purine nucleoside of a target sequence of a
single stranded nucl~ic acid, a corresponding nucleoside
of a Second Strand and a corresponding nucleoside of a
Third Stand wherein the Second Strand nucleoside comprises
a pyrimidine analog which has a Watson-Crick binding face
capable of binding by Watson-Crick base pairing to the
i
lS
21~96~
WO94tll~34 PCT/US93/1117B
purine base and a Second-Third Strand binding face having
at least two hydrogen binding sites. The purine nucleo-
side of the target sequence is contacted with the Second
Strand nucleoside and a Third Strand nucleoside which has
a base complementary to the Second-Third Strand binding
face of the base of the Second Strand nucleoside to give
a triplet.
Preferred target sequences for detection, recognition
and/or inhibition or alteration of expression by the
Second and Third Strands according to the methods of the
present invention have from about 4 to about 40 nucleo-
sides. Sequences of this length are long enough to be
unique, but are short enough for selectivity towards the
target sequence (the Second and Third Strands are unlikely
to bind to an unrelated target sequence).
:
Definitions
As used herein, the following terms have the
following meanings unless expressly stated to the
contrary.
:: : :
The term "purine" or ~"purine base'l includes not only
the naturally occurring adenine and guanine bases, but
also modifications of ~ those bases such as bases
substituted at the 8-position, or guanine analogs modified
at the 6-position or the analog of adenine, 2-amino
purine, as well as analog~ of purines having carbon
replacing nitrogen at the 9-position such as the 9-dea~a
; purine deri~atives and other~purine analogs such as those
set forth in Figure 4 herein.
The term "nucleoside" includes a nucleosidyl unit and
is used interchangeably therewith, and refers to a subunit
of a nucleic acid which comprises a 5-carbon sugar and a
- ~ nitrogen-containing base. The term includes not only
hose nucleosidyl units having A, G, C, T and U as their
bases, but also analogs and modified forms of the
naturally-occurring bases, including the pyrimidine-5-
donor/acceptor bases such are pseudoisocytosine and
: .
2i919626
WO94/1153~ PCT/~S93/1117~
pseudouracil and other modified bases (such as 8-
substituted purines). In RNA, the 5-carbon sugar is
ribose; in DNA, it is a 2'-deoxyribose. The term
nucleoside also includes other analogs of such subunits,
including those which have modified sugars such as 2'-O-
alkyl ribose.
For con~istency and in order to avoid confusion we
are employing an alternative numbering system for the
rings of the pyrimidine and purine analogs used herein so
that the number assigned to a ring position will be the
:: : same relative to the position on the ring of the C-C or N-
C glycosidic bond between the base or base analog and
: sugar without regard to whether a nualeoside is a C- or a
N- nucleoside and without regard to the position of ring
nitrogens. This numbering system is based on .the
:~ numbering used for the naturally occurring pyrimidine and
purine N-nucleosides. In the context of the pseudo (~)
pyrimidine C-nucleoside, it may be called the "pseudo'l (or
") numberin~ system, or alternati~ely just the number of
thé ring position may be used. T~e numbering system is
shown in figures 3A and 3B wherein each X may be
independently nitrogen or carbon.
O .:~
~5 ~
:The term 'iphosphonate" refers to the group O=P-R
` I .
wherein R is hydrogen or;~an alkyl or aryl group. Suitable
alkyl or aryl groups include those which do not sterically .
hinder the phosphonate linkage or interact with each
other. The phosphonate group may exist in either an "R"
: o.r an "S" configuration. Phosphonate groups may be used
as internucleosidyl phosphorus group linkages (or links)
: to connect nucleosidyl units.
. .
, :
: `
2149~26
WO94/llS34 PCT/US93/11178
. The term "phosphodiester'l refers to the group O=P-O
o
. ' I
. wherein phosphodiester groups may be used as
; internucleosidyl phosphorus group linkages (or links) to
connect.nucleosidyI units.
A "non-nucleoside monomeric unit~ refers to a
monomeric unit wherein the base, the sugar and/or the
phosphorus backbone has been replaced by other chemical
moieties.
A "nucleoside/non-nucleoside polymer" refers to a
polymer comprised of nucleoside and non-nucleoside
monomeric u~its~.:
The term !'oligonucleoside" or "Oligomer" refers to a
chain of nucleosides which are linked by internucleoside
:linkages which is generally from about 4 to about l00
nucleosides in length,~but which may be greater than about
100 nucleosides in length. They a~b usually synthesized
from nucleoside~ monomers, but may also be obtained by
enzymatic means. Thus, the term "Oligomer" refers to a
chain of oligonucleosides: which have internucleosidyl
;~ linkages linking the nucleoside monomers and, thus,
includes oligonucleotidesJ nonionic oligonucleoside alkyl-
and a~yl-pho~phonat ;analogs, alkyl- and aryl-
. phosphonothioates, phosphorothioate or phosphorodithioate
' : 30 analogs of oligonucleotides~ phosphoramidate analogs of
oligonucleotides, neutral phosphate ester oligonucleoside
analogs, such as phosphotriesters and other
: oligo~ucleoside analogs and:modified oligonucleosides, and
also includes nucleoside/~on-nucleoside polymers. The
3:5 term also includes nucleoside/nucleotide polymers wherein
: one or more of the phosphorus group linkages between
` monomeric units has been replaced by a non-phosphorous
linkage such as a formacetal linkage, a sulfamate linkage,
or a carbamate linkage. It also includes nucleoside/non-
r5
.~A~
2149~26 -~
094/11534 PCTJUS93/11178
nucleoside polymers wherein both the sugar and the
phosphorous moiety have been replaced or modified such as
morpholino base analogs, or polyamide base analogs. It
also includes nucléoside/non-nucleoside polymers wherein
the base, the sugar, and the phosphate backbone of the
non-nucleoside are either replaced by a non-nucleoside
moiety or wherein a non-nucleoside moiety is inserted into
the nucleoside/non-nucleoside polymer. Optionally, said
non-nucleoside moiety may serve to link other small
molecules which may interact with target sequences or
alter uptake into target cells.
The term '!alkyl- or aryl-phosphonate Oligomer" refers
to Oligomers having at least one~ alkyl- or aryl-
phosphonate internucleosidyl linkage. Suitable alkyl- or
~; 15 ~aryl- phosphonate groups include alkyl- or aryl- groups
which do not sterical~ly hinder the phosphonate linkage or
interact with each other. Preferred alkyl groups include
lower alkyl groups~ having from about l to about 6 carbon
atoms. Suitable~aryl groups have~at least one ring ha~ing~ ~ 2~ a conjugated pi electron system an~ incIude carbocyclic
aryl and heterocyclic aryl groups, which may be optionally
substituted and preferabIy having up to about l0 carbon
atoms.
: ::
The ~ term~ "methylphosphonate Oligomer" (or "MP-
Oligomer") refers to Oligomers having at least onemethylphosphonate internucleosidyl linkage.
The term "neutral Oligomern refers to Oligomers which
have nonionic internucleosidyl linkages between nucleoside
monomers (i.e. r ~linkages having no positive or negative
ionic charge) and include, for example, Oligomers having
, . ~ , ...
internucleosidyl linkages such as alkyl- or aryl-
phosphonate linkages r alkyl- or aryl-phosphonothioates,
neutral phosphate ester linkages such as phosphotriester
linkages, especially neutral ethyltriester linkages; and
non-phosphorus-containing internucleosidyl linkages, such
as sulfamate, morpholino, formacetal, and carbamate
linkages. Optionally, a neutral Oligomer may comprise a
W094/11534 2149~26- PCT/US93/11178 ~ I
14
conjugate between an oligonucleoside or nucleoside/non- i
nucleoside polymer and a second molecule which comprises
a conjugation partner. Such conjugation partners may
comprise intercalators, alkylating agents, binding
substances for~cell surface receptors, lipophilic agents,
nucleic acid modifying groups including photo-cross-
linking agents such as psoralen and groups capable of
cleaving a targe~ted portion of a nucleic acid, and the
like. Such conjugation partners may further enhance the
uptake of the Oligomer, modify the interaction of thé
~1 Oligomer wlth the target~ sequence, or alter the
pharmacokinetic distribution of the Oligomer. The
essential requirement is ~that the oligonucleoside or
; nuc;leoside/non-nucleoside polymer that the Oligomer
conjugate comprlses be neutral.
The term "substantially neutral Oligomer" refers to
Oligomers in which at least about 80 percent of the
int~ernucleosidyl linkages between the nucleoside monomers
are nonionic linkages.
The term "neutral alkyl- o~ aryl- phosphonate
Oligomer" refers to~ neutral Oligomers having neutral
, .
~ internucleosidyl linkages which comprise at least one
.
alkyl- or aryl~ phosphonate linkage.
The term "neutral methylphosphonate Oligomer" refers
to neutral Oligomers having~ internucleosidyl linkages
which comprise at least one methylphosphonate linkage.
The term "complementary," when referring to Second
, and Third Strands, referq to Strands having base sequences
which allow the Strand or Oligomer to hydrogen bond with
~; 30 the~base sequence-of the target sequence of a nucleic acid
or another strand and thus bind to the nucleic acid or
1 other strand and in combination to form a triple helix
, ~ ~
complex.
In the various Oligomer sequences listed herein, "p"
as listed in ~pG represents a phosphodiester internucleo-
1~ `
side linkage and ~ as in C~G represents a methylphospho-
nate internucleoside linkage. Also the notation such as
;......................................... 2149626
WO94/11534 PCT/US93/11178
T indicates nucleosides linked by methylphosphonate
linkages.
The term "triplet" or "triad" refers a hydrogen
bonded complex of three nucleoside bases ~etween a base of
a target sequence, a base of a first Oligomer and a base
of Oligomer as set forth in one of motifs I to V' of
Figure l.
Brief Description of the_Drawinqs
Figure l depicts triplet formation motifs using the
Watson-~rick pairs of permutation l of Fiyure 2A.
Figure ZA depicts four possible permutations of
Second Strand bases for use in recognizing the! naturally
occurring bases of the target sequence. These Second
Strand bases have at least two hydrogen bonding positions
lS on their Second-Third~Strand binding faces so as to be
able to selecti~ely hydrogen bond with a base of a Third
Strand nucleoside.
Figure 2B depicts motifs I to V' for selection of
Third Strand bases dependent on the hydrogen bonding
pattern of the Second-Third Strand binding face of the
Second Strand base.
Figure 3A depicts the numbering system used herein
for pyri~midine and pyrimidine analog bases. Figure 3B
depicts the ring numbering system used herein for purine
and purine analog bases.
Figure 4 depicts the structures of and abbreviation
for certain bases used according to the methods of the
present invention to form triple helix complexes.
Figure S depicts the Watson-Crick base paring schemes
for permutation 1 of Figures 2A.
Figure 6 depicts the Watson-Crick base pairing
schemes for permutation 4 of Figure 2A.
Figure 7 depicts triads formed with the Watson-Crick
base pairs of permutation l according to motif I.
Figure 8 depicts triads formed with the Watson-Crick
base pairs of permutation l according to motif I'.
. -
.,
2l49~2~ j ~
WO94/11534 PCT/US93/lil78
16
Figure 9 de~icts triads formed with the Watson-Crick
base pairs of permutation l according to motif II.
Figure lO depicts triads formed with the Watson-Crick
base pairs of permutation ~ according to motif II'. . -
Figure ll depicts triads formed with the Watson-Crick
base pairs of permutation l according to motif III.
Figure 12 depicts triads formed with the Watson-Crick
base pairs of permutation l according to motif III'.
Figure 12 depicts triads formed with the Watson-Crick
base pairs of permutation 1 according to motif IV.
Figure 14 depicts triads formed with the Watson-Crick
base pairs of permutation l ~ccording to motif IV'.
Figure 15 depicts triads formed with the Watson-Crick
~ase pairs of permutation l according to motif V.
Figure 16 depicts triads formed with the Watson-Crick
base pairs of permutation l accordlng to motif V'.
'
Detailed Descri tion of the Invention -~
General StrateqY
I The antisense strategy for the~development of speci- -~
1 20 fically synthesized oliyonucleotides (and their analogs)-
as sequence-specific/gene-specific therapeutic agents, has 1-
~,
now become a major direction for drug development. Two
conflicting/contradictory ~demands in the design of the
Second and Third Strands are of primary concern. The
therapeutic Second and Third Strands should be absolutely
sequence-specific for their designated target sequences in
order to avoid unwanted interactions with the large number
~ of other nucleic acid sequences within the cells. Adven-
; ~ titious interactions of administered Oligomers with other
sequences could lead to undesirable side effects. In
addition, there is a requirement of high affinlty of the
Oligomers for the target, so that low concentrations for
the Oligomers will be sufficient for the masking function
at the target site. These two considerations are related
to each other in that if the Oligomer's binding to the
target nucleic acid is very specific and with high
,
6 2 S
i ~ W~94/i1534 ' P~T~S93~11178 l;
'i ,
17
affinity, then the concentration of Oligomer needed for
the desired therapeutic effect are reduced and th~e side
effects may become non-existent. This high specificity
and affinity is important, especially for systemic
treatment when the entire body of the ~atient is treated
with the Oligomers. Additionally, high affinity is
~; significant in that minimum amounts of Oligomers will be
' required to produce the desired effect, thereby reducing
~ the cost of treating the patient.
i 10Mechanistically, howe~er, high affinity and high
3:~: specificity are contradictory requirements. In kinetic
terms, high af~inity requires a very slow or non-existent
'off-rate once Oligomer and the target are bound to each
other. However, a sufficientl~ high off-rate must exist
ln order for each Oligomer to search and to determine if
the site to which~it is bound is completely complementary
to its sequencej hence ensuring that the bound site is the
correct target ~ite.~ Thus,~ in order for each Oligomer to
be highly specific, it must have the ability to search and
to determine its complementarity o~ the interacting site
,1t~; for a perfect match, properties which require a fast off-
rate. On the other hand, for the binding to have a high
affinity, the O1igomer and the orrect'target complex with
a perfect match must have a very slow off-rate. In all of
these processes, the on-rate should be controlled by
; dlffusion and will be influenced by the accessibility of
the target sequence'of nucleic acid to the Oligomers and
only slightly by the size (number of monomeric units) of
the 'Qligomers.
30In addition to the strictly chemical and thermo-
dynamic considerations, the kinetic element in living
processes is a very vital concern. All of the reactions
and interactions in living cells are related to each other
functionally in a kinetic manner, and are not necessarily
related to each other'in a thermodynamic manner. If an
i~ inappropriate binding exists too long because of an
1~ insufficient off-rate, then damage to the cell may have
1 ,; .
2 1 ~
WO 94/1 ~534 PCr/USg3/1 1 17~ Ç~ i~
.
18
sufficient time to occur, thus leading to undeslrable
biological effects. Therefore, on one hand, we need to
have a rapid process of search and determination during
the initial phase of interaction, but, on the other hand,
we need a very slow process of dissociation once the
l correct binding between the probe and the target nucleic
j acid occurs.
The above description leads to the next conclusion
that for a successful antisense strategy, the interaction
between the Oligomers and the target nucleic acid should
be a two-step process. The first step is "Search". The
major objective in this step is for the Oligomers to
rapidly screen interactions with all of the possible
nucleic acid targets inside the living cells and tissues
as quickly as possible, with a relatively fast off-ra e.
After the successful search, which leads to a proper
interaction of the Oligomers and the single-stranded
target nucleic~acid (usually RNA, but it can also be DNA),
I ~ , .
l~ a second step is now required for the "Sealing", leading
~ 20 to the formation of a complex whic~ has a very slow off-
¦~ rate. One demonstration of that strategy is in our
publications and patent applications which describe utili-
zing a psoralen derivatized OIigomer. See, e.q., United
States Serial No. 06/924,234 and published PCT Application
No. WO 92/02641. In this case, the Oligomer is reasonably
short, 10-12 nucleotides in length, and has been deriva-
tized with psoralen which is a photo-reactive crosslinking
group. Upon photo-irradiation the psoralen on the
Oligomer can form a cyclobutane-type of crosslinking with
a double bond in a pyrimidine base, for exampIe, cytosine
or uracil located in the target strand only in a perfectly - i
matched duplex. Since the Oligomer will be covalently
linked to the target nucleic acid in the perfectly matched .
duplex upon photo-irradiation, the off-ratP is now
~;~ 35 practically reduced to zero for the covalent complex.
~ 2149625
'~ WO94~ 34 PCT/US93/11178
19 .
The challenge is to be able to form a similar type of
complex in two steps, but to eliminate the re~uirement for
an external energy source, such as photo-irradiation.
Tri~le Helix Complex Formation With the Tarqet Nucleic
Acids
Formation of triple-stranded helices, triple helix
complexes or triplexes, in nucleic acid physical chemistry
has been reported where a Watson-Crick type of pyrimi-
dine:purine duplex has a pyrimidine third strand bound in
10 its major groove using Hoogsteen-type base pairing as the ~-
motif for the base triad. The most well known case is the
T-A-T}/ (or U-A=U) base triad, as well as the C-G=C+
triad. In this situation, T-A=T or U~A=U can be formed at
neutral pH, or without additional contribution by proto-
nation. On the other hand, in the system of C~G=C+,
~;protonation on the third strand of C is required. There-
fore, the formation of such triplexes is sensitive to pH
changes around neutrality. We have determlned alternative
compounds which can be used in plac~ of C for triple helix
-formation which has eliminated this requixe~ent (proto-
nation of C).~ One such compound is the pseudo-isocytosine
nucleoside. With these alternative bases, an appropriate
hydrogen-bonding site is provided in the neutral unproto-
nated base for triple helix formation. The use o pseudo-
~5 isocytosine in the third strand to form triple helixcomplexes is described in our co-pending application,
United States Sèri;al No. 07/772,081.
In that application, we have outlined two possible
arrangements for triple helix construction with the target
~;30 nucleic acid as one strand, and with two Oligomers as the
Second and Third Strands in the triple helix complex, the
"closed sandwich" and the "open sandwich". The "closed
In T~A-T, "-" refers to the Watson Crick base pairing
between the single stranded targPt (in bold) and Second Strand
3~r taken together double stranded target) and "-" refers to
the palring within the Third Strand.
.4g62~
WO94/l1~34 PCT/US93~11178 ~ .
.
,~."i
~ 20
'li .
sandwich" arrangement can be formed when the target
sequence consists only of purine residues and involves the
binding of a homopyrimidine Oligomer as a Second Strand as
~,ja Watson-Crick complement to one side of the target strand
(at the C6, N1, C2 face of the purine base) and another
Oligomer as a Third Strand binding to the other side of
the target strand's purine bases which offer two hydrogen-
bonding sites (the C6, N7 face) in the major groove. In
this case, each Oligomer participates in sequence specific
hydrogen-bonding with the target strand and the OIigomers
~(Second and Third Strands) do not participate in hydrogen-
i~3ibonding with each other; i.e. the target sequence is
enclosed by hydrogen-bonding interactions with the Oli-
gomers. This type of arrangement is termed a closed
triplex or "clo~sed sandwich" because the target sequence
is sandwiched between two Oligomers. The !'open sandwich"
arrangement using naturally occurring bases can be formed
when the target sequence consists only of pyrimidine
residues. Here sequence specific Watson-Crick inter-
actions are satisfied by a homop~rine Oligomer (SecondStrand). However, in order for triple helix formation to
occur, thisi Second Strand must interact with a Third
Strand at its C6, N7 face.~ In this case only the Second
Strand makes sequence specific hydrogen-bonds with the
:s:
'~25 target sequence and the Second and Third Strands share a
~hydrogen-bonding interface and hydrogen bond with each
`3;other. In this option, the target strand is on an open
9~ j ~side of the trlple helix complex. This second type of
~arrangement is termed an open triple helix or "open
;~30 sandwich".
From theoretical considerations involving short
nucleic acid target sequences, it is possible that for
dissociation of the target nucleic acid, after triple
helix formation, so that it is completely free of inter-
actions with uncomplexed Oligomers, a closed sandwich maybe a more favorable arrangement than an open sandwich.
This understanding is somewhat intuitive as the target
. :
2149626
WO 94/11534 PCIJ~JS93/11178 i.
21
strand must break away from two sets of hydrogen-bonding
interactions with the Oligomers in the closed sandwich
case, whereas there is only one such set of interactions
to break in the open sandwich case. However, this consi-
5 deration may not be relevant when the target nucleic acid
is a large molecule with only a small segment of the
target sequence in a single-stranded form, and, thereby,
available for sequence-specific complex formation. For
such a large molecule plus small Oligomer interaction, the
10 entropy considerations strongly favor dissociation of the
complex due to the departure of the small Oligomer from
the larger nucleic acid molecule. In this case, the
closed sandwich may not have any advantage ove~r the open
sandwich, and may even be more sterically hindered than
15 the open sandwich. Therefore, for the complex formation
between a large target nucleic acid and small Oligomers,
the open sandwich arrangement may be preferred. In this
l case, the Second Stxand is bound as the Watson-Crick
f complement to the target sequence and is restricted from
20 dissociating by the added Third Str~hd. This Third Strand
binding-may decrease the dissociation constant of the
complex by 100 to 1000 fold, and could reduce the needed
concentration for therapeutic action, for instance, from
100 ~M to 1.0 or 0.1 ~M. More importantly, the length
2~ requirement for the available open sequence of the target
nucleic acid can still be relatively short, such as from
10 to 14 nucleotide units. The target nucleic acid is
much more likely to have an open single-stranded region of
such a length, instead of a longer (2 20 nucleotides)
30 sequence. ~fff
. .
~; ~ Additional theoretical considerations for not using
long Second and Third Strands are described in Ts'o, et
al., ~nnual, NY Academy of Sciences, in press ~to be
published 1992). ;~
'' ' . '.
.
2 1 ~ 9 6 2 ~ 1.
i WO94~11534 PCT/US93/11178 ~ ¦
,
I
! 22
Sequence Restr _tion in Triple Hellx Formation
The reported strategies involved in triple helix
formation at specific target sites, and the ability to
have a workable antisense therapeutic application through
triple helix formation, has been greatly limited by the
requirement of the homopurine or homopyrimidine sequence.
3~ Simply stated, until now we were unable to form stable
;
sequence-specific triple helixes without having the
single-stranded nucleic acid target consisting of only
purines or only pyrimidines. The present invention
provides a breakthrough related to this restriction, i.e.,
triple helix complexes may be formed with any sequence
arrangement of the single-stranded nucleic acid target.
The major limitation in the triple helix formation
has been that the~ pyrimidine in the Watson-Crick duplex
has only one additional hydrogen bonding site after the
formation of the duplex via Watson-Crick hydrogen bonding
scheme. One aspect of the present invention is to use C-
nucleoiides for the pyrimidines in the Second Strand of
the Watson-Crick duplex formed with ~he target sequence in
replacement of the naturally occurring N-nucleoside. With
~ the C nucleosides, the glycosidic ~ond between the
-~ pyrimidine base and the sugar moiety is a carbon-carbon
bond, whereas with the N-nucleosides, the pyrimidine base
and sugar are~attached by a nitrogen-carbon bond. In such
a manner, the C-pyrimidine nucleoside has an additional
hydrogen bonding site for a pair of hydrogen bond
;~ ~ formation with the third st~rand added ;to the Watson-Crick
duplex. Since there is only a small change in the C-C
bond vs. the C-N bond distance (about 0.lA), the original
nucleic acid structure is preserved with minimal
perturbation. In this manner, the usefulness of C-
pyrimidine nucleosides is greatly magnified for a triple
helix formation as compared to the naturally occurring N-
pyrimidine nucleoside.
Accordingly, the present invention provides a compre-
i ~ .
1~ hensive approach for triple helix formation with target
I ~
214962~
~ W094i11534 PCT/US93/11178
.
~,~
23
''!~q nucleic acid sequences consisting of any combination of the four naturally occurring bases which is described
below. The bases of some of the nucleosides proposed for
;j use in the Second ~nd Third Strands are naturally occurr-
ing minor bases, such as pseudo-uracil, and xanthine and
are commercially available; syntheses for other of the
unusual bases have appeared in the literaturei and yet
other may be prepared by syntheses analogous to literature
syntheses. (See included references yiven in "Nucleoside
~f~ 10 Bases" herein below). Figures 7 to 16 depict triads
formed according to motifs I to V' of Figure l (or
~ ~ Permutation l of Figure 2A).
:.................................................
Second Strand - Pre~er ed Pyrimidlne-5-Donor/Acceptor
ases
'~ 15As noted the Second Strand incorporates in place of
the naturally oc~urring cytidine or uridine (or thymidine)
~; nucleosides, analogs of these nucleosides which are able
j~3 to form Watson-Crick base pairs with the target sequence,
but also have an additional hydrog~n bonding site at the
position which corresponds to the 5-position of cytidine
or uridine, which we ha~e termed pyri~idine-5-donor/
acceptor bases nucleosides.
Preferred pyrimidine-5-donor/acceptor bases nucleo-
sides include C-nucleosides, that is where the glycosidic
bond is attached to a carbon atom of the heterocyclic
base, rather than to a ring nitrogen. Attachment to a
carbon atom allows the ring nitrogen to be available for
hydrogen bonding.
The proposed hydrogen bonding patterns and isomorphic
geometries requires the use of several nonstandard (i.e.
not naturally occurring) heterocyclic bases. We have
adopted a numbering format for all the nucleosides based
on the numbering format used for the naturally occurring
purine and pyrimidine nucleosides. The atom numbering for
the n~turally occurring purine and pyrimidine nucleosides
is set forth in Figures 3A and 3B. For nucleosides with
1:
~J
1 i
`'`''
n ~
2l49626~ ! -
W094/115~ - PCT/US93/11178
24
an N-glycosidic linkage to the sugar, the atom numbering
follows that for the standard bases. For these bases the
covalent attachment to the sugar is at the N9 position for
purines and at Nl for pyrimidines. Under conventional
numbering guidelines the numbering for the C-nucleosides
in relation to the glycosidic bond would be different due
to the different positioning of the heterocyclic nitrogens
in relation to the glycosidic bond. In order to avoid
confusion due to these differences in numbering, we are
employing an alternative numbering system for the C-
nucleosides (~-pyrimidines) in this application ~$ee
Figure 3A). This "pseudo" numbering system allows the
position of hydrogen bonding sites on the C-nucleosides to
be analogous to the standard bases in relatlon to the
glycosidic bond. Thus, pseudouridine will have hydrogen
bonding acceptors at the ~02 and ~04 positions. Note that
this hydrogen bonding pattern is the same as for the
stan~ard uridine nucleoside except for the additional
donor site at the ~N5 position.
Four different permutations of ~he four Watson-Crick
type base pairs which form the basis of the triple helix
formation motifs are depicted in Figures 2A and 2B. The
.
binding motifs using preferred permutation l are further
tabulated in Figure l. In Figure l, target sequence and
Second Strand bases in each pair are on the left and
right, respectively, with glycosidic linkages and the ~-
minor groove oriented downward. Strand polarity is
indicated at the Cl' position. ~ -
For purposes of these figures, the ~arget strand base ' ~-
will be indicated first in bold with the complementary
base in the Second Strand separated by a bullet (-~ - -
representing Watson-Crick hydrogen bonding schemes in each
pair. Recognition of target sequence pyrimidine bases by - -
;~ Second Strand bases gives the standard base pairs C~G and
U-A. According to a preferred embodiment, recognition of
target sequence purine bases is accomplished by C-
nucleoside pyrimidine bases on the Second Strand to give
:'
~'
O i = ~ .. .,, ., . . , . . , . . .. . ~ . . . . . . . ... . . . . . .... . . ... . . .
`' ~W094/ltS34 ~1~9~2S pC~/US93/11178
,., . t
.~ 1
~5
the base pairs, ~iC and A~U. The hydrogen bonding
- pattern for these "pseudo Watson-Crick" base pairs is the
~ same as ~or the standard GC and A-U base pairs. However,
^~ by use of these pyrimidine-5-donor!acceptor bases in the
Second Strand there is a pair of hydrogen bonding
.~i donor/acceptor sites in the major groove of the double
-.~ helix at the pyrimidine base on the Second Strand, an
;~ additional site for hydrogen bonding is provided at ~N5 of
each C-nucleoside at a position approximately isomorphous
10 to N7 of the purines in the C~G and UA base pairs. The
:;
~; four base pairing schemes (target ~ Second Strand) each
have a unique pattern o hydrogen bond donors and
acceptors on the Second Strand on the back side facing the
major groove (i.e., on the Second-Third Strand binding
` 15 face). Specifically, C4G has two acceptors, Go~iC has two
donors, UA has a donor and an acceptor (as viewed from
~ ~ ~ ~ the major groove) and A~U has an acceptor and a donor.
x ~ ; Use of these unique patterns of hydrogen bonding sites on
; ~ the;bases of the Second Strand for the four target bases
s ~ ~0 make it possible to construct a ser~es of isomorphic base
~ ~ triad motifs.
;, ~ ,
Third Strand Bas _Selection
Selection of nucleosides (or bases) or the Third
.
Strand may be based on one of triad motifs I to V' of
25 Figures 1 and 2B. These motifs are based upon Third
Stxand recognition by either pyrimidine or purine
nucleosides and are separated into thr e classes according
~; to their general recognition schemes. Syste~atic con-
struction and ordering of these motifs will be according
30 to the following set of guidelines. First, it is assumed
that all ~ucleosides on the Third Strand will have the
anti configuration of the base at the glycosidic linkage.
Therefore, proposed Third Strand polarities can be made
directly by comparison to the Watson-Crick strands.
35 Second, a pair of specific hydrogen-bonds must be made to
the Watson-Crick Second Strand by adjacent donor/acceptor
. ~
wo 94,ll~342 1 4 9 6 2 6 PCT/US93/11178 ~
26
sites on the -Third Strand base. As discussed in detail
below, pyrimidine bases possess two sets of adjacent sites
(C4~N3 and N3-C2) whereas purine bases have three (C6-Nl,
Nl-C2 and C6-N7). Third, the overall form of the base
triads should be geometrically isomorphous. Because the
target strand may contain a heterologous sequence of
bases, the Watson-Crick section of the base triads will
have the familiar pseudo dyad symmetry of the base pair.
However, due to the differences in shape of the pyrimidine
and purine bases, the third~strand may be only of one type
of base (i.e.,~ all pyrimidine or all purine). This
requirement is important to the formation of triple-
stranded helices in order to ensure regular positioning of
the Third Strand backbone and to optimize stacking
i~teractions between adjacent triads.
Motifs I, I', II and II' (Class A motifs) are
constructed using pyrimidine Third Strand bases. Motif I
is based upon the most well known base triad, T-A=T
(analogous to U~A=T). Here the Third Strand T (at 04 and
H3) accepts and donates a hydrogen~bond to A (at H6 and
N7~. This type of hydrogen-bonding scheme was first
identified by Hoogsteen in crystals of l-methylthymine and
; ~ ~ 9-methyladenine. The strand polaritie~ of triple helices
containing only this triad has been shown to be anti-
parallel for the~Watson-Crick interaction and parallel for
the Hoogsteen interaction. The isomorphic triads in this
motif are generated by ident1fying pyrimidine bases which
possess the requisite hydrogen-bond donor/acceptor pairs
at C4 and N3 or at comparable position in ~-pyrimidines.
Recognition of the Second Strand at G requires two donors
on the Third Strand. Thisi is provided by the ~iC base
(note that the tautomeric form is now different than that
required for Watson-Crick recognition by this base) or
~; alternatively the ~iC base. The following discussion is
directed to permutation l of Figure 2A which is more fully
depicted in Figure l. The individual triads of motifs I
to V' for permutation l are depicted in Figures 7 to 16.
2149625
. ~ 094/11534 PCT/US93/111f8
;t
~, .
27
Recognition of Second Strand ~iC requires two acceptors
provided by the natural base analog iC The triad
completing motif I involves recognition of ~U by C which
provides a donor and acceptor (again, as viewed from the
~:; 5 major groove of the Watson-Crick helix). Motif I' is
related to motif I by an inversion of the polarity of the
Third Strand backbone, maintaining Third Strand
it~ recognition by donor-acceptor sites at C4 and N3 positions
s, of the Third Strand pyrimidine bases. Therefore, base
pairing in~ol~ing two donors to two acceptors (C-G~iC or
~: G~iC~iC) can be constructed by flipping the orientation
~:; of the base ~thereby the orientation of the backbone) for
mot1f I 180 in the plane of the paper so that the
donor/acceptor at N3 (or ~N3) is now hydrogen-bonding at
. 15 the site on the Second Strand base closest to the major
:~ groove and the substituent at C4 (or ~C4) is now hydrogen-
bondlng toward~the minor groove. Such a rotation of the
bases in motif I for the UA=T or A-~U=C triads will
result in mispairing. However, an interchange of the
: 20 Third Strand bases allows the co~rect hydrogen bonding
~: ; patterns to be made, resulting in the triads ~A=C and-
A~U~T for moti~ I'. (See Figure 8). Construction of the
triads ~or motif II involves recognition of the Second
~ Strand base by N3 and C2 of the Third Stand pyrimidine
j~ 25 bases. As fcr motif I, the Third Strand is parallel to
: the strand to which it binds. The base triads CG-~C,
~: G~iC=C, U~A=iC and A~U=T as shown in Figure 9 are
proposed. Motif II' is. related to motif II by similar
rules interconverting motifs I and I'. Here it should be
noted that the following triad of motif II':UoA=T involves
~ an A=T hydrogen bonding scheme of the type also found in
l~ ~ crystals of thymine and adenine known as reverse
Hoogsteen. (See Figure lO).
~ It should be noted -that the proposed base triad
i~ 35 motifs include a subset which can be utilized to recognize
~ naturally occurring double-stranded target pyrimidine-
j~ purine sequences. The base pairs C-G and T~A found in DNA
t~9~5:
'~ WO 94/1 1534 PCT/VS93/11178 ~f
~ ~d
`i 28
,~,lj~ .
:~ are equivalent to the third and fourth Watson-Crick pairs
in each moti (See Figure 1). Therefore, it may be
possible to form triple-stranded helices at double-
stranded target sites by the addition of a single oligomer
probe designed accordiny to the rules of the ten motifs
presented here. For example, it was described in United
,~
States Serial No. 07/772,081, filed October 7, 1991 of
, ~ ~ which the present application is a continuation-in-part,
that a synthetic oligonucleotide probe containing ~iC and
U residues may bind sequence specifically to a homopyrimi-
dine~homopurine target sequence, forming a triple-stranded
complex according to motif I. Other examples of pyr-
pur-pur triple helices formed from homopolymer sequences
have been reported. The C-G=G triple helix first reported
by Lipsett, M.N., J. Biol. Chem. 239:1256-1260 (1963), may
form according to either motif IV or IV', although the
~; recent studies on intramoleculair complexes, which forces
the two purine strands to be antiparallel, seems to
indicate that motif IV' is the preferred pur=pur
interaction.
In general,~base substitutions may be made in any-
motif as long as the specific hydrogen bonding patterns
are maintained and the new triad remains isomorphic to the
remaining triads in the motif. For instance, Third Strand
recognition by purine bases in motifs III, III', IV and
IV~ does not involve~hydrogen-bonding at N7. (See, e.a.,
Figures 11 to 14). There~ore, it may be advantageous to
synthesize some or all of~the Third Strand residues as 7-
deaza- analogs (derivatives of tubercidin) in order to
avoid unwanted interactions at this face of the Third
Strand. In addition, the su~ar moiety and backbone
linkages of the Oligomer probe strands can be any that are
available. The choice of these elements for the backbone
should be made based upon their ability to confer chemical
~-~ 35 stability and favorable characteristics in terms of
binding stability and specificity. Obviously, a number of
choices are available regarding both sugar and backbone
~; 214962~:
WO9~ 34 PCT/US93/ll178
29
linkage. Common sugar moieties include 2'-deoxyribose,
ribose, or 2'-O methylribose. Suitable backbones for the
Third Strand include phosphodiester, methylphosphonate or
phosphorothioate.
...
B. Figures 2A and 2B
The binding moti~s set ~orth in Figure l plus three
other permutations of Second Strand recognition schemes
having Watson-Crick complementarity to the four naturally
~ occurring target strand bases and also having unique
;~ lO nydrogen bonding patterns on their Second-Third Strand
binding faces for Third Strand recognition are set forth
in Figure 2A.
In Figure 2B, the hydrogen bonding patterns for the
Second-Third Strand binding face are depicted by the
15 double arrow or pair of arrows to the right of the Second
Strand base. For Third ~Stand base recognition using two
hydrogen bonds, four hydrogen bonding patterns are
possible. The~se patterns are indicated as follows: ~
represents two donor sites, ~ represents two acceptor
20 sites, z represents a donor and an acceptor site in a
specific orientation, and~ ~represents an acceptor and a
donor slte in a specific orientation. The triads for
motifs I to V' ~using Second Strand bases selected
~; according to permutation l of Figure 2A and Third Strand
25 ~ases selected according to motifs I to V' are depicted in
Figures 7 to 16.
Figure 2B depicts the Third Strand binding motifs for
each of the four specified Second-Third Strand binding
patterns. Strand polarity of target, Second and Third
30 strand are indicated in the right hand column. E
Base sequences for appropriate Second and Third -
Strands to form a triple helix complex with a target 3
strand having any combination of pyrimidine nucleosides
~; may be conveniently determined using Figures 2A and 2B.
:
` 2149626 -
WO94/11~34 PCr/US93/11178 ~ :
.
~ ;,
Second and Third Strands
The Second and Third Strands may comprise separate
Oligomers. Alternatively, the Second and Third Strands
may be covalently linked together.
Preferably the Second and Third Strands each comprise
from about 4 to about 40 nucleosides, more preferably from
about 6 to about 30 nucleosides and especially preferred
are Second and Third Strands of about 8 to about 20
nucleosldes.
A. Internucleoide Linkaqes
The Second and Third Strands of the present invention
comprise optionally covalently linked Oligomers.
Oligomers having the desired internucleoside linkages
~; may be conveniently prepared according to synthetic
techniques known to those skilled in the art. For
example, commercial machines, reagents and protocols are
available for the synthesis of Oligomers having
phosphodiester and certain other phosphorus-containing
internucleoside linkages. See also Gait, M.J.,
Oliqonucleotide Synthesis: A _Practical Approach ~IRL
Press, 1984); Cohen, Jack S., Oliqodeoxynucleotides
Antisense Inhibitors of Gene Expression, ICRC Press, Boca
Raton, FL, 1989); and Oli~qonucleotides and Analoques: A
Practical ~pproach, (F. Eckstein, ed., l99l). Preparation
of Oligomers having certain non-phosphorus-containing
internucleoside linkages is described in United States
Patent No. 5,142,047, the disclosure of which is
incorporated herein by reference.
. Nucleoside Bases
71 30 As set forth herein, the Second and Third Strands of
; the present invention may include certai~ analogues of the
naturally occurring pyrimidine and purine bases. These
analogs incIude the above-noted pyrimidine-5-
~,~ , .,
3~~ ~ donor/acceptor bases. The synthesis of these bases used
~ 35 in our proposed binding motifs have been reported and by
,~ ~
: ` l
,,~ 21~g626 -
~ WO94/11534 PCT/US93/11178
., .
31
following those literature procedures, those bases can be
made.
The ring structures for some of these bases and their
abbreviations are set forth in Figure 4.
5In particular, the following bases may be prepared
according to the following reported procedures.
The synthesis of pseudoisocytidine (~iC) is reported
by Ono, A., et al., J. Org. Chem. 57:3225-3230 (1992).
The synthesis of 5-aza-cytidine (5aC or
pseudoisocytidine~ or ~iC~) is reported by Beisler, J.A.,
; et al., J. Carbohyd.;Nucleosides Nucleotides 4:281-299
(197?)-
Pseudouridine~ (~U) is commercially available ~from
Kyowa Hakko Kogyo Co. Ltd., N.Y.).
15The synthesis of pseudocytidine is reported by
Pankiewlcz, K.W., et al., Carbohyd. Res. 127:227-233
(1984)~.
The syntheses of~9-deaza-guanosine (9deaZaG or 9daG) and
i sine (9deaZaI or 9daI) are reported by Lim, et al.,
20J. Org. Chem. 48:780-788 (1983).
The synthesis of 9-deaza~adenosine (9dea~aA or 9daA) iS
reported by~Lim and Klein, Tetrahedron Letters 22:25-28
1981). ~ ~
~ The synthesis of isocytidine (iC) is reported by
;~ 25Switzer, C., et~ al., J. Am. Chem. Soc. 111:8322-8323
89).
~ Isoguanosine~ (IG~)~ is synthesized by methods
¦ j analogous to those reported by Revanker et al., J. Med.
~hem. 27:1389 (1984) for 3-deazaguanine.
~ Inosine (I) an~ its ~phosphoramidite synthon are
commerci~lly available (from Cruachem, Herndon, VA).
Inosine~ (I ) and isoinosine' (iI) may be prepared by
methods analogous to those reported by Rosemeyer and
; Seela, J. Org. Chem. 52:5136-5143 (1987) for 5-aza-7-
;35 deazaguanosine.
~ Xanthine (X) and Xanthosine are commercially
s~ ~ ~available (from Sigma).
~}~
W094/llS34 2 1 ~ 9 6 2 6 PCT/US93/11178 ~ i~
.
32
The synthesis of 2-amino-purine (2ap) is reported by
McLaughlin, L.W., et al., Nucl. Acids Res. l6:563l-5644
(ls88) and by Doudna, J.A., et al., J. Org. Chem. 55:5547-
5549 (1990~. '
"
S Second and Third Strand Complementarity
;~ Preferred are Second and Third Strands that each have
a corresponding nucleoside complementary to each
nucleoside o~ the target sequence (i.e., have "exact
complementarity"). However, included within the scope of
the present invention are Second and Third Strands which
may lack a complemen~ for each nucleoside in the target
~equence, provided that the Second Strand has such binding
affinity for the target sequence and the Third Strand has
;~ ~ ; sufficient binding affinity for the Second Strand that
together the Second and Third Strands bind with the target
sequence to recognize it or to inhibit its expression by
forming a triple helix complex. Such strands are referred
- to as being "substantially complementary" or having
"substantial complementarity".
The Second Strand should be substantially comple-
mentary to the target sequence and the Third Strand should
;~ ~ be substantially complementary to the Second Strand in
that there is sufficient hybridization and hydrogen-
bonding between the strands for inhibition of expression
of the target sequence, and if the target sequence is a
portion of a mRNA, Inhibition of translation, to occur.
Sufficient hybridization~and hydro~en-bonding is related
to the strength of the hydrogen-bonding between bases as
well as the specificlty of the complementary strand. The
strength of the~ hydrogen-bonding is in~luenced by the
~ number and percentage of bases in a strand that are base
-~ paired to complementary bases, according to either Watson-
Crick base pairing (for target-Second Strand bindingj or
between Second Strand and Third Strand, whether by
previously described triplet formation schemes or by one
of triad motifs I to V'. To be specific, the co~plemen-
` ~ ~ ' ' I
~ ~'WO94~ 34 ' 2149 62-6 PCT-/US93/1117~ ~
, ~
i:~
,~ 33
.~
tary bases of the strand must be sufficient in number so
as to avoid non-specific binding to other sequences within
a genome and while at the same time small enough in number
to avoid non-specific binding between other sequences
within a genome and portions of a long strand.
It will also be appreciated that the base sequence o~
~, either the Second or the Third Strand need not be lO0
,,'~ percent complementary to the sequence to which it is to
i~ bind. Preferably the sequence is at least about 80
percent complementary, more preferably at least about 90
percent and eve,n more,preferably about 95 percent or more.
The Second and Third Strand may optionally include one or
more non-nucleoside monomeric units. Such non-nucleoside
monomeric units include those described in co-pending U.S.
Serial No. 07/565,307, filed August 9, l990 (also pub-
lished PCT Application No. WO g2/02532), the disclosure of
3 ~ ~ which is incorporated herein by reference. The strand in
question need only be capable of sufficient hybridization
or bonding to the target sequence (or the Second Strand)
,~1 20 to prevent or interfere with expr~ssion of the target
sequence, such as by pre~enting normal translation of the
target sequence or to specifically recognize the target
se~uence. Pre~ention of normal translation of the target
sequence occurs when an expression product of the target
sequence lS produced in an amount significantly lower than
would be ~he result in the absence of the Second and Third
Strands. The expression product is a protein. Measure-
ment ,of the decrease in production o~ proteins is well
,-~ known to those skllled in the art and such methods include
~uantification by chromatography, biological assay or
immunological reactivity.
Utility and Administration
J~ According to the present invention, a specific seg-
ment of single stranded nucleic acid may be detected or
35~ recognized using Second and Third Strands which form ~
triple helix with the single stranded nucleic acid
.. ~ , ~.
2149626
W094/11~34 PCTJUS93/111i8
34
according to the triplet base pairing guidelines described
herein. The Second and Third Strands have sequences
selected as described above such that a base of the Second
Strand will hydrogen bond with a base of the target
sequence (by its Watson-Crick binding face) and with a
corresponding base of the Third Strand (by its Second-
Third Strand binding face? to give a triplet and, thus, to
result in a triple helix comple~. The Second and Third
Strands are Oligomers which may be optionally covalently
linked. Detectably labeled Oligomers may be used as
proved for use in hybridization assays, for example, to
detect the presence of a particular single-stranded
nucleic acid sequence.
The present invention also provides a method of
preventing or altering expression or function of a
selected target sequence of single stranded nucleic acid
by use of Second and Third Strands which form a triple
stranded helix structure with the single stranded target
as described above. Formation of the triple stranded
helix may prevent expression and/or~unction by modes such
as preventing transcription, preventing of binding of
effector molecules (such as proteins), etc.
According to the methods of ~he present invenkion, a
high affinity-complex îs formed with a high degree of
~5 selectivity. Derivatized Second and Third Strands may be
used to detect or locate and then irreversibly modify the
target site in the nucleic acid by cross-linking
(psoralens) or cleavin~ one or both strands (EDTA). By
careful selection of a target site for cleavage, one of
the strands may be used as a molecular scissors to
specifically excise a selected nucleic acid sequence.
The Second or Third Strands may be derivatized to
incorporate a nucleic acid reacting or modifying group
-which can be caused to react with the nucleic acid segment
or a target sequence thereof to irreversibly modify,
degrade or destroy the nucleic acid and thus irreversibly
inhibit its functions.
.
~.~ 21~96~6
~ ~ 094/11534 PCT~US93/11178
}
;,~ These Second and Third Strands may be used to
~1 inactivate or inhibit or alter expression of a particular
i,~ gene or target sequence of the same in a living cell,
allowing selective inactivation or inhibition or
alteration of expression. The target sequence may be DNA
or RNA, such as a pre-mRNA, an mRNA or an RNA sequence
such as an initiator codon, a polyadenylation region, an
mRN~ cap site or a splice junction. These strands could
then be used to permanently inactivate, turn off or
, 10 destroy genes which produced defective or undesired
products or if activated caused undesirable effects.
-Another aspect of the present invention is directed
to a kit for detecting a particular single stranded
nucleic acid sequence which comprises Second and Third
Strands at least one of which is detectably labeled and
selected to be able sufficiently complementary to the
target sequence of the~single stranded nucleic acid to be
able to form a triple helix structure therewith.
Since the Second and Third Strands for use with the
methods of the present inventio~ form triple helix
complexes or other forms of stable association with
Y~ transcribed regions, these complexes are useful in
~ "antisense" therapy~ "Antisense" therapy as used herein
J ;~ iS a generic term which includes the use of specific
binding Oligomers to inactivate undesirable DNA or RNA
sequence~ ln vitro or _ vlvo.
Many diseases and other conditions are characterized
by the presence of undesired~DNA or RNA, which may be in
certain instances single stranded form and in other
0 instances in double stranded form. These diseases and
conditions can be treated using the principles of
antisense therapy as is generally understood in the art.
Antisense therapy includes target~ing a specific DNA or RNA
target sequence through complementarity or through any
other specific binding means, in the case of the present
invention by formation of triple helix complexes according
to the binding motifs described herein.
~: .
~ 2 1 4 9 6 2 ~
~ W894/11~34 PCT/US93/11178 ~
,
iS1 36
,;
The Oligomers for use in the instant invention may be
administered singly, or combinations of Oligomers may be
~ administered for adjacent or distant targets or for
;~ combined effects of antisense mechanisms with the
l 5 foregoing general mechanisms.
`~ In therapeutic applications, the Oligomers can be
~, formulated for a ~ariety of modes of administration,
,~ including sy~temic, topical or localized administration.
Techni~ues and formulations generally may be found in
10 Rem1nqton's Pharmaceut1cal Sclences, Mack Publishing Co.,
3 Easton, PA, latest edition. The Oligomer active
ingredient is generally combined with a carrier such as a
diluent or excipient which may include fillers, extenders,
binding, wetting agents, disintegrants, surface-active
- 15 agents, or lubricants, depending on the nature of the mode
of administration and dosage forms. Typical dosa~e forms
include tablets, powders, liquid preparations including
suspensions, emulsions and solutions, granules, capsules
and suppositories, as well as liquid preparations for
20 injections, including lipos~me prep~rations.
For systemic administration, injection may be
preferred, including intramuscular, intravenous,
intraperitoneal, and subcutaneous. For injection, the
Oligomers for use with the invention are formulated in
25 liquid solutions, preferably in physiologically compatible
buffers such as Hank's solution or Ringer's solution. In
addition, the Oligomers may be formulated in solid form
and redissolyed or suspended immediately prior to use.
~yophilized forms are also included.
~; 30 Systemic administration can also be by transmucosal
or transdermal means, or the compounds can be administered
orally. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are
used in the formulation. Such penetrant~ are generally
3S known in the art, and include, for example, bile salts and
~ fusidic acid derivatives for transmucusal administration.
'~t!~: In addition, detergents may be used to facilitate
7:
.,
`i
~ WO94/11~34 214~fi26 Pcr/USg3/11178
37
permeation. Transmucosal administration may be through
use of nasal sprays, for example, as well as formulations
suitable for administration by inhalation, or
suppositories. For oral administration, the Oligomers are
formulated into conventional oral administration forms
such as capsules, tablets, and tonics.
For topical administration, the Oligomers for use in
the invention are formulated int~ ointments, salves, eye
drops, gels, or creams, as is generally known in the art.
lOIn addition to use in therapy, the methods of the
- present invention may be used diagnostically to detect the
presence or absence of the target DNA or RNA sequences to
which the Oligomers specifically bind. Such diaynostic
tests are conducted by hybridization throuyh triple helix
complex formation which is then detected by conventional
-
~;~ means. For example, Oligomers may be labeled using
radioactive, fluorescent, or chromogenic labels and the
presence of label bound to solid support detected.
Alternatively, the presence of a triple helix may be
20 detected by antibodies which specifically recognize forms. -~
Mean~ for conducting assays using such Oligomers as probes
are generalIy known. `
-.
.
..
-:
. .
,.,
..
~ .
