Note: Descriptions are shown in the official language in which they were submitted.
~094/15620 2 1 $ 3 ~ 5 7 PCT~S94/00585
NOVEL OLIGONUCLEOTIDES MODIFIED WITH NON-NUCLEOTIDE
BRIDGING GROUPS
This invention relates to oligonucleotide duplexes.
More particularly, this invention relateJ to &ingle or
double-stranded oligonucleotides in which the 5' and 3' end~
of opposing oligonucleotide strand~ are linked by novel
~ridging groups.
The synthe~is of single or double-stranded
oligonucleotides with natural nucleotide bridging groups has
been deJcribed in the literature. Erie, et al.,
Biochemistry, Vol. 28, 268-273 (1989) describe the synthe~is
of a 26-residue double-stranded circular oligonucleotide
using five thymidylate re~idues for each bridging group.
Formation of the closed circular form wa~ carried out using
T4 DNA liga~e. A~hley, et al., BiochemistrY~ Vol. 30, pgs.
2927-2933 (1991), di~close the con~truction of cyclic
oligonucleotide~, or dumbbells, which employ four thymidyllc
acid r~Jidue~ a~ the bridging group~. Cyclization of the
linear ollgonucleotide precursor wa~ achieved using a
carbodiimidc as the coupling agent. Amaratunga, et al.,
Biopolymer~, Vol. 32, pgq. 865-879 (1992), diJclo~e the
preparation of S~tJ of double-~tranded oligonucleotides
having 16 b~se pair~ in which the number of thymidylate
bridging group6 wa~ varied between 2 and 14. These
oligonucleotideJ were also circularized u~ing an enzymatic
W094/15620 ~53~ -2- PCT~S94tO058~
method. Single-Stranded circular oligonucleotides have also
been d~cribed in Kool, J. Am. Chem. Soc., Vol. 113, pg~.
6265-6266 (1991), by Prakash, et al., J. Chem. Soc. Chem.
Commun., pgs. 1161-1163 (1991), and by Prakash, et al., J.
Am. Chem. Soc., Vol. 114, pgs. 3523-3527 (1992).
Chu, et al., Nucl. Acids Res., Vol. 19, pg. 6958,
disclo~e the binding of hairpin and dumbbell DNA sequences
to transcription factors. These DNA sequence~ employed
natural nucleotide~ for the bridging group~.
Durand, et al., Nucl. Acids Re~., Vol. 18, pgs.
6353-6359 (1991) dlsclo~e self-complementary
oligonucleotides containing a hairpin loop, or bridging
group. The loop consists of a hexaethylene glycol chain,
and the oligonucleotide could form a hairpin structure a~
effectively a~ the analogous oligonucleotide possessing
thymidylate re~idues for the bridging group~.
Glick, et al., J. Am Chem. Soc., Vol. 114, pgs,
5447-544B (1992), di~close the formation of a
double-stranded oligonucleotide which is cros~linked through
linker arms attached to the ba~es rather than the sugar
phosphate backbone.
In accordance with an a~pect of the pr~ent invention,
there is provided an oligonucleotide having a structural
formula ~elected from the group consi~ting of:
51 ' I - S,~_
Xl X2 and X1 X~2
L
52 54 S5
Sl, S2, 53, S4, and S5 are oligonucleotlde strands.
Each of X1 and X2 is a nucleotide ~trand, such as a
nucleotide bridging loop, or a non-nucleotide bridging
W094/15620 ~ S 3 0~ 7 PCT~S94/00~85
moiety. Each of X1 and X2 may be the same or different, and
when one of X1 and X2 is a nucleotide strand, the other of
X1 and X2 i8 a non-nucleotide bridging moiety. Thus, each
of X1 and X2 independently is a bridging moiety ha~lns flrst
and second termini that each bind~ independently with a
nucleotide phosphate moiety or a nucleotide hydroxyl moiety
Examples of terminal moieties that bind with phosphate
moieties; e.g., terminal phosphates of nucleic acid
sequences, include, but are not limited to, -OH groups, -NH2
groups, and -SH groups.
Examples of terminal moieties that bind with a hydroxyl
moiety, particularly the terminal hydroxyL moiety of a
nucleic acid ~equence (e.g., the ribose of a 3' terminal
nucleotide), including, but are not limited to, _po32
group~, -SO3 groups, and -COO groups.
When Xl and/or X2 is a non-nucleotide bridging moiety,
the non-nucleotide bridging moiety may have the following
structural formula:
Tl - ~ - T2, whereas each of T1 and T2 independently
binds with a nucleotide pho~phate moiety or a hydroxyl
moiety. R i~ ~elected from the group con~isting of (a)
~aturated and un~aturated hydrocarbons; (b) polyalkylene
glycols; (c) polypeptides; (d) thiohydrocarbons; (e)
polyalkylamine~; tf) polyalkylene thioglycols; (g)
polyamide~; (h) dicub~titute~ monocyclic or polycyclic
aromatic hydrocarbons; (i) intercalatinq agents; (j)
mono~accharidea; and ~k) oligo~accharides; or mixtures
thereof.
In one embodiment, one of Tl and T2 binds with a
nucleotide pho~phate moiety, and the other of T1 and T2
binds with a nucleotide hydroxyl moiety.
W094/15620 2 1 S 3 ~ S 7 PCT~S94/00585
In one embodiment, the oligonucleotide has the
structural formula:
- - Sl-
Xl X2
S
In one embodiment, at least a portion of S1 is
complementary to S2. In another embodiment, all of S1 and
52 are complementary to each other ~uch that Sl and 52 bind
to form a double-stranded region.
In yet another embodiment, 51 is not complementary to
52~ and the oligonucleotide molecule exists a~ an unpaired
oligonucleotide.
In another embodiment, the oligonucleotide has the
structural formula:
S3
Xl X2
s4 S5
In one embodiment, at least a portion of 53 is
complementary to portions of S4 and/or S5. In another
embodiment, all of 54 and S5 are complementary to 53 such
that S4 ~nd S~ bind to 53 to form double-~tranded regions.
In yet another embodiment, S3 is not complementary to
S4 and S5, and formation of double-stranded regions is not
possible, ~o that the oligonucleotide molecule exists as an
unpaired oligonucleotide.
The term "ollgonucleotide" as used herein means that
the oligonucleotide may be a ribonucleotide,
deoxyribonucleotide, or a mixed
W094/lS620 21 S3D~ 7 PCT~S94/00585
ribonucleotide/deoxyribonucleotide; i.e., the
oligonucleotide may include ribo~e or deoxyribose sugars or
both. Alternatively, the oligonucleotide may include other
5-carbon or 6-carbon sugars, such as, for example arabinose,
xylose, glucose, galactose, or deoxy deri~atives thereof or
any mixture of sugars.
In one embodiment, each of Sl, and S2, or S3, and 54
and S5 combined may include from about 5 to about lOO
nucleotide units, and preferably from about lO to about lO0
nucleotide units.
The phosphorus containing moieties of the
oligonucleotide may be, for example, a phosphate,
phosphonate, alkylphosphonate, aminoalkyl phosphonate,
thiophosphonate, phosphoramidate, phosphordiamidate
phosphorothioate, phosphorothionate, phosphorothiolate,
phosphoramidothiolate, and pho~phorimidate. It i~ to be
understood, however, the scope of the present invention is
not to be limited to any specific phosphorus moiety or
moieties. The pho~phorus moiety be modified with cationic,
anionic, or zwitterionic moieties. The oligonucleotides may
also contain backbone linkages which do not contain
phosphorus, such as carbonates, carboxymethyl e~ter~,
acetamidates, carbamates, acetals, and the like.
The oligonucleotides may include any natural or
unnatural, Jub~tltuted or unsubstituted, purine or
pyrimidine ba~e. Such purine and pyrimidine bases include,
but are not limited to, natural purines and pyrimidines,
such a~ adenine, cytosine, thymine, guanine, uracil, or
other purines and pyrimidines, such as isocytosine,
6-methyluracil, 4, 6-di-hydroxypyrimidine, hypoxanthine,
xanthine, 2, 6-diaminopurine, 5-azacyto~ine, 5-methyl
cytosine and the like.
In one embodiment, each of X1 and X2 i~ a
non-nucleotide bridging moiety having the formula T1-R-T2,
W094/15620 PCT~S94/00585
2153~57 -6-
as hereinabove de8cribed. In one embodiment, R i8 a
~aturated or un~aturated hydrocarbon, and preferably R is a
polyalkylene moiety wherein the polyalkylene group has from
5 to lOO carbon atoms, preferably from 5 to 20 carbon atoms.
Most preferably, the polyalkylene is a polymethylene moiety
The bridging moiety including the polyalkylene group may be
attached to the sugar phosphate backbone of the
oligonucleotide.
In another embodiment, R i8 a polyalkylene glycol. In
particular, the polyalXylene glycol has the structural
formula (R~O)n, wherein R is an alkylene group having from 2
to 6 carbon atoms, preferably from 2 to 3 carbon atom~, and
n is from 1 to 50, preferably from 3 to 6. In one
embodiment, the polyalkylene glycol i~ polyethylene glycol,
and preferably the polyethylene glycol is he~aethylene
glycol.
Bridging moieties including polyalkylene glycols may be
attached to the oligonucleotide by converting the
polyalkylene glycol into a material which may be employed in
a DNA ~ynthesizer. For example, the polyalkylene glycol may
be converted to its mono-dimethoxytrityl ether, which is
then reacted with chloro-N, N-dii~opropylamino-
cyanoetho~y-pho~phine to produce a bridging group
phosphoramiditc. For oligonucleotide synthe~is, a
pho~phorylating agent is attached to a solld ~upport of a
DNA ~ynthe~izer, and a series of DNA ba~e~ i~ delivered in
order, dc ~ ing upon the sequence required for binding to
the target DNA, RNA, protein or peptide. The bridging group
phosphoramidite i8 then added, followed by the addition of a
further sequence of DNA bases. Optionally, another bridging
group is then attached, and a further sequence of DNA bases
is added to complete the oligonucleotide ~equence.
At the conclu~ion of the synthe~is, the oligonucleotlde
is cleaved from the ~olid support wlth ammonia to give a
WO94/15620 ~57 PCT~S94/00585
-7-
crude trityl-containins oligonucleotide pos~e~sing a
3'-pho~phate group. Purification i8 carried out u~ing
rever~ed pha~e HPLC and the later eluting, trityl-containing
oligonucleotide i8 collected. The oligonucleotide is
detritylated ucing acetic acid, extracted with ethyl acetate
to remove trityl alcohol, and lyophilized to give an
oligonucleotide which can hybridize to it~elf to form an
open chain 3'-phocphorylated oligonucleotide. Reaction of
thi~ open chain oligonucleotide with a carbodiimide coupling
agent in an aqueous buffer produces a clo~ed circular
oligonucleotide. In one procedure, small portion~ of the
carbodiimide coupling agent are added at infreguent
interval~ a~ de~cribed in Example 2. In another procedure,
a large exces~ of coupling agent is added at the beginning
of the procedure as described in Exumple 4.
Unpaired open chain oligonucleotide~ can be
circularized using a coupling agent such a~ cyanogen bromide
in the pre~ence of a complementary oligonucleotide ~plint as
described by Praka~h, et al., J. Chem. Soc. Chem. Commun.,
pgs. 1161-1163 (1991) or using a water ~oluble carbodiimide
in the pre~ence of a complementary oligonucleotlde ~plint as
described by Dolinnaya, et al, Nucl. ACidc Re~., Vol. 16,
pgc 3721-373B (1988).
In another embodiment, R i~ a polypeptide.
Polypeptid-- whlch can be included in the bridging groups
include, but are not limited to, hydrophobic poly~peptides
such a~ (Ala)n, basic polypeptides Cuch a~ (Lysn), and
ac~dic polypeptldes ~uch as (Glu)n, wherein n ic from 3 to
50, preferably from 4 to 10. In an alternative embodiment,
the polypeptideo may contain mixture~ of amino acids.
Such bridging groups including polypeptides may be
attached to the oligonucleotide by procedures such as that
given in Example 7 hereinbelow.
WO94/15620 PCT~S94/00585
215~7
In yet another embodiment, R is a poLyalkylamine.
Polyalkylamine~ which may be included in the bridging groups
include, but are not limited to tho9e having the following
structural formula:
R3NHl(cH2)m NHRl]p ~ [(CH2)n MHR2lq
wherein each of Rl, R2, and R3 is hydrogen or an alkyl group
having from 2 to lO carbon atoms, and wherein m and n are
from 2 to lO, preferably from 2 to 4, and p and q each are
from 2 to 20, preferably from 3 to 6. Rl, R2 and R3 may be
the same or different, m and n can be the ~ame or different,
and p and q can be the ~ame or different. Examples of such
polyamines include polyethylene imine, which has the formula
H2N(CH2 CH2 NH)rH, wherein m and n each are 2, p+q=r, and
Rl, R2, and R3 are H; and spermidine, which has the
2 ( H2)4 NH-(CH2)3-NH2, wherein m i~ 4, n is 3 p
is l, and q i 8 l, and each of Rl, R2, and R3 i~ hydrogen
Ex~mples of polyalkylene thioglycols which may be
included in the bridging groups include, but are not limited
to,
3,6 dithio-l,8-octanediol,HOCH2CH2SCH2CH25CH2CH2OH,
2-mercaptoethyl sulfide, (HSCH2CH2)2S,
3,3'-thiodipropanol, S(CH2CH2CH2OH)2,
2-mercaptoethyl ether, (HSCH2CH2)2O, and
2,2'-dithiodlethanol S(CH2CH2OH)2.
E~ample~ of thiohydrocarbons such as polyalkylene disulfides
which may be employed ln the bridging groups include, but
are not limited to, 2-hydroxyethyl di~ulfide (HOCH2CH2)252
An example of incorporation of one of these moietie~ as a
bridging group i~ described in Example 9 hereinbelow.
In another embodiment, R i~ a polyamide.
Polyamides which may be included in the bridging group~
include tho~e having the following structural formula:
~094/15620 1S30S~ PCT~594/0~585
H~NH(CH2) -CO1 OH
wherein m i8 from l to 6, and n is from 3 to 50.
Bridging groups containing such polyamides may be
attached to the oligonucleotide by procedures such as those
given in Example 7 hereinbelow.
In yet another embodiment, R is a disubstituted
monocyclic aromatic. Disubstituted monocyclic aromatics
which may be included in the bridging groups include those
having the following structural formula:
(CH2)m(-x ~ Y)n-(CH2)m, wherein each of X and Y
is -CONH, or X is -NHCO and Y is COMH, m is from l to lO and
n is from l to 5. In another embodiment,-the disubstituted
monocyclic aromatic may have the following structural
formula:
( O X)n, wherein X i5 oxygen, sulfur, or -OCH2,
and n is from l to lO.
In another embodiment, R is a disubstituted polycyclic
aromatic hydrocarbon. Disubstituted polycyclic aromatic
hydrocarbon~ which may be employed include, but are not
limited to, di~ubstituted naphthalenes, anthracenes,
phenanthrenes, fluorenes, and pyrene~. Bridging moieties
containing di~ubstituted aromatics may be attached to the
oligonucleotide by procedures such as given in Example lO
hereinbelow.
In yet another embodiment, R i~ an intercalating agent.
Intercalating agent~ which can be included in the
bridging moieties include, but are not limited to,
acridine~, phenanthrid~nes, anthracyclinones, phenazines,
phenothiazine~, ~nd qu~nollnes. Bridging moieties including
such agents may be attached to the oligonucleotide by
procedure~ such as those given in Example ll hereinbelow.
In another embodiment, R is a monosaccharide, or in yet
another embodiment, R is a oligosaccharide. Monosaccharides
and oligosaccharide~ which may be included in the bridging
W094/15620 PCT~S94/00585
~1S3~7 -lo-
group~ include, but are not limited to, glucose, mannoce,
and galactose; disaccharides such as cellobiose and
gentobio~e; trisaccharides such a~ cellotriose; and larger
oligosaccharides such as cellotetraose, cellopentaose, and
pentamannose.
Bridging moieties containing such monosaccharides and
oligosaccharide~ may be attached to the oligonucleotide by
procedures such as those given in Example 12 hereinbelow.
The oligonucleotides of the present invention may be
employed to bind to RNA ~equences by Watson-Crick
hybridization, and thereby block RNA proce~sing or
translation. For example, the oligonucleotide~ of the
present invention may be employed as "antisense" complements
to target ~e~uences of mRNA in order to effect translation
arrest and selectively regulate protein production.
The oligonucleotides of the pre~ent invention may be
employed to bind RNA or DNA to form triplexe~, or triple
helice~. Single stranded oligonucleotide~ have been
described to bind double-stranded DNA and thereby interfere
with transcription in Maher, et al., 8iochemistrY, Vol. 29,
pgs. B820-8826 (1990) and in Orson et al., Nucleic Acids
ReE ., Vol. 19, pgs. 3435-3441 (1991).
Similarly, ~uch triplex formation could be expected to
interfere with replication. SingLe-stranded
oligonucleotide~ could also be envisaged to bind to
double-ctranded (e.g., viral) RNA to form triplexes which
block transcription or reverse transcription. Circular
paired oligonucleotides may be employed to form "rever~e
triplexes" in which the paired oligonucleotides form
triplexe~ with a ~ingle-stranded RNA or DNA target, thereby
blocking transcription, replication or reverse transcriptior
of said RNA or DNA target.
Triple helix formation by oligonucleotides in a
~equence-~pecific manner is normally re~tricted to
O 94/1~620 ~S PCT/llS94/00585
?
polypurine tract9 of duplex DNA. In order to increase the
number of targets for triple helix formation, Froehler et
al., in BiochemistrY, Vol. 31, pg~. 1603-1609 (1992) and
Horne, et al., in J. Am. Chem. Soc., Vol. 112, pgs.
2435-2437 (1990) utilized oligonucleotides containing a
3',3' internucleotide junction or linker group to allow for
bindlng to opposite strands of DNA. Unpaired circular
oliqonucleotides of the pre~ent invention can be employed to
form "~witchover" complexe~ with double-stranded DNA or RNA
as shown in the following ~tructure:
nucleic acid target
oligonucleotide
In such complexes, pyrimidines on one portion of the
bridged cyclic oligonucleotide interact via Hoogsteen
interaction~ with purine~ on one strand of the nucleic acid
target, while pyrimidines on another portion of the
oligonucleotide interact with purines on the other strand of
the target. Structure~ of this type do not need an
interveninq linker group and have the added advantage that
stacking interactions are maintained. The bridging residues
and the cyclic nature of the oliqonucleotide serves to
minimize degradation by nuclea~es. Unpaired cyclic
oligonucleotide~ forminq switchover comple~e~ as described
herein can be envi~aged to occur with target double-stranded
WO94/15620 215 3 ~ 5 ~ PCT~S94/00~85
DNA or RNA, thereby blocking transcription, replication, or
rever~e transcription.
The paired or unpaired circular oligonucleotides of the
pre~ent invention may be employed to bind specifically to
target protein~, or to selected regions of target proteins
80 a~ to block function or to restore functions that had
been lost by a protein a~ a result of mutation. For
example, the oligonucleotide~ of the pre~ent invention may
be used to block the interaction between a receptor and its
ligand(s) or to interfere with the binding of an enzyme to
its sub~trate or cofactor or to interfere otherwise with the
catalytic action of an enzyme. Convercely, the
oligonucleotides of the pre~ent invention may be employed to
restore lost function to a mutated protein, for example, by
eliciting conformational alteration of such a protein
through formation of a complex with that protein.
In another embodiment, the oligonucleo'ide~ may bind to
tran~criptional activators or suppre~sors. Such factors
might, for example, enhance transcription of cellular DNA,
in order to regulate cellular gene expression. As a further
example, the oligonucleotide~ may inhibit the action of the
protein encoded by the mvb oncogene, which act~ as a
tran~criptional activator (Gabriel~en, et al., Science, Vol
253, pgs. 1140-1143 (1991)). When this protcin is
inappropriately expressed, it can activate genes leading to
the formatlon of a cancer. 8inding of the myb protein to
the oligonucleotide~ of the pre~ent invention would block
the gene activation and block the growth of the cancerous
cell~.
Alternatively, the oligonucleotides may bind to viral
transcription factors. For example, the oligonucleotides
may inhlbit human immunodeficlency (HIV) tran~criptional
activatorr or enhancer~ or bovine or human papilloma virus
tran~criptional activators or enhancer~. Alternatively, the
WO94/15620 1 S3 ~ ~ 7 PCT~S94/00585
-l3-
oligonucleotide~ may activate gene expression by binding to
and preventing activity of, transcriptional repressors.
Bielinska, et al., Science, Vol. 250, pgs. 997-lOOO
(November 16, l99O), disclose double-stranded
phosphorothioate oligonucleotides which bind to
transcription factors or enhancers of viru~es such as HIV
Such oligonucleotides may also be added to Jurkat leukeml a
T- cells in order to inhlbit interleukin-2 secretion.
Androphy, et al., Nature, Vol. 325, pgs. 70-73 (January 1,
1987), disclose a 23 ba~e pair oligonucleotide which
prevents binding of the E2 protein of bovine papilloma virus
(BPV) to the upstream regulatory region of the BPV genome,
which immediately precedes the early genes of the 8PV
genome. European Patent Application No. 302,758 discloses
double-stranded oligonucleotide~ which bind to transcription
enhancers of bovine papilloma virus or human papilloma
virus, thereby repressing the transcription of the DNA of
the virus and lnhibiting the growth of the viru~. The above
oligonucleotides disclosed in the above-mentioned
publications may be modified to include the bridging
moieties of the pre~ent invention and still be employed for
binding to transcription factors or en~an~er~.
The RNA, DNA, protein or peptide target of interest, tc
which the oligonucleotide binds, may be present in or on a
prokaryotic or eukaryotic cell, a virus, a normal cell, or a
neoplastic cell, in a bodi!y fluid or in stool. The target
nucleic acid~ or proteins may be of plasmid, viral,
chromo~omal, mitochondrial or plastid origin. The target
sequence~ may include DNA or RNA open reading frames
encoding proteins, mRNA, ribosomal RNA, JnRNA, hnRNA,
introns, or untranslated 5'- and 3'-sequences flanking DNA
or RNA open readlng frames. The modified oligonucleotide
may therefore be involved in inhibiting production or
function of a particular gene by inhibiting the express~on
W094/15620 PCT~S94/00~85
~I53~7 -14-
of a repressor, enhancing or promoting the function of a
particular mutated or modified protein by eliciting a
conformational change in that protein, or the modified
oligonucleotide may be involved in reducing the
proliferation of viruses, microorganisms, or neoplastic
cells. The oligonucleotides may al~o target a DNA origin of
replication or à rever~e tranqcription initiation site.
The oligonucleotide~ may be u~ed in vitro or in vivo
for modifying the phenotype of cells, or for limitinq the
proliferation of pathoqens such as viruses, bacteria,
protists, Mycoplasma species, Chlamydia or the like, or for
killing or interfering with the growth of neoplastic cells
or specific classes of normal cells. Thus, the
oligonucleotideR may be administered to a host subject in a
diseased or susceptible state to inhibit the transcription
and/or expre~sion of the native genes of a target cell, or
to inhibit function of a protein in that cell. Therefore,
the oligonucleotides may be used for protection from, or
treatment of, a variety of pathogens in a host, such as, for
example, enterotoxigenic bacteria, Pneumococci, Neisseria
organism~, Giardia organisms, or Entamoebas, etc. Such
oligonucleotides may also inhibit function, maturation, or
proliferation of neoplastic cells, such as carcinoma cells,
sarcoma cells, and lymphoma cells; specific B-cell~;
~pecific T-cell~, such as helper cells, suppre~sor cells,
cytoto~ic T-lymphocytes (CTL), natural killer (NK) cells,
etc.
The oligonucleotide~ may be selected ~o as to be
capable of interfering with RNA processing (transcription
product maturation) or production of proteins by any of the
mechanisms involved with the binding of the subject
composition to its target ~equence. These mechanisms may
include interference with processing, inhibition of
W094/15620 215 3 ~ ~ 7 PCT~S94/0058~
- 1 5 -
transport across the nuclear membrane, cleavage by
endonucleases, or the like.
The unpaired, circular oligonucleotides may contain
sequence~ complementary to those present in growth factors,
lymphokines, immunoglobulins, T-cell receptor sites, MHC
antigens, DNA or RNA polymerases, antibiotic resistance,
multiple drug resistance (mdr), genes involved with
metabolic proces~es, in the formation of amino acids,
nucleic acids, or the like, DHFR, etc. as well as introns or
flankinq sequence~ a~sociated with the open reading frames.
The following table is illustrative of some additional
applications of the subject compositions.
Area of Application Specific APplication Targets
Infectious Disea~es:
Antivirals, Hum-n HIV, HSV, CMV, HPV, VZV
infections
Antivirals, Animal Chicken Infectious Bronchitis
Pig Transmissible
Gastroenteritis Virus
infections
Antibacterial, Human Drug Resistance Plasmids
Antipara~itic Agent~ Malaria
Sleeping Sicknes~
(Trypanosomes)
Cancer
Direct Anti-Tumor Oncogenes and their products
Agent~ Tumor Suppre~sor genes and their
products
Adjunctive T~erapy Drug Resistance genes
and their products
W094/15620 PCT~S94/00585
215305~ -16-
Auto Immune Diseases
T-cell receptors or Rheumatoid Arthritis
autoantibodie~ Type I Diabetes
Systemic Lupus
Multiple 5C lerosi 8
Organ Transplants OKT3 cells causing
GVHD
The oligonucleotides of the present invention may be
employed for binding to target molecules, such a8, for
example, proteins including, but not limited to, li~ands,
receptors, and or enzymes, whereby such oligonucleotide~
inhibit the activity of the target molecules, or restore
activity lost through mutation or modification of the tar~et
molecules.
The oliyonucleotides of the present invention are
adminlstered in an effective binding amount to an RNA, a
DNA, a protein, or a peptide. Preferably, the
oligonucleotides are administered to a host, such a~ a human
or non-human animal host, 80 as to obtain a concentration of
oligonucleotide in the blood of from about 0.1 to about 100
~mole/l. It i~ also contemplated that the oligonucleotides
may be admini~tered in vitro or ex vivo a~ well as in vivo
The oligonucleotide~ may be admini~tcred ln coniunction
with an acceptable pharmaceutical carrier a~ a
pharmaceutical composition. Such pharmaceutical
composition~ may contain suitable excipients and auxiliaries
which facilitate processin~ of the active compound~ into
preparations which can be u~ed pharmaceutically. Such
oligonucleotide~ may be administered by intramuscular,
intraperitoneal, intraveneou~, or ~ubdermal injection in a
quitable ~olution. Preferably, the preparation~,
particularly those which can be administered orally and
~0 94/15620 21 S3 o ~ 7 PCT/US94/00585
--17-- ~
which can be used for the preferred type of administration,
such as tablet~, drageeQ and capsules, and preparations
which can be administered rectally, such as suppositorie~,
as well as ~uitable 601utions for administration
parenterally or orally, and compositions which can be
administered ~ ~c ally or sublingually, including inclusion
compounds, ccnr~-_n from about 0.1 to 99 percent by weight of
active ingredients, together with the excipient. It is also
contemplated that the oligonucleotides may be administered
topically in a suitable carrier, emul~ion, or cream, or by
aerosol.
The pharmaceutical preparation~ of the present
invention are manufactured in a manner which is itself well
known in the art. For example, the pharmaceutical
preparation~ may be made by mean~ of conventional mixing,
granulating, dragee-making, di~olving or lyophilizing
processes. The process to be used will depend ultimately on
the physical properties of the active ingredient u~ed.
Suitable excipients are, in particular, fillers such as
sugar, for example, lactose or sucro~e, mannitol or
sorbitol, cellulo~e preparations and/or calcium phosphates,
for example, tricalcium phosphate or calcium hydrogen
phosphate, as well as binders such as starch or paste,
using, for e~cample, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydro~cypropylmethylcellulose, sodium carboxypropylmethyl-
cel~ulo~e, ~odium carboxymethylcellulo~e, and/or polyvinyl
pyrrolidone. If desired, disintegrating agents may be
added, ~uch a~ the above-mentioned starche~ a~ well as
carboxymethyl-~tarch, cross-linked polyvinyl pyrrolidone,
agar, or alginic acid or a salt thereof, such as sodium
alginate. Auxiliaries are flow-regulatlng agents and
lubricants, such as, for example, silica, talc, ~tearic acid
or salts thereof, such as magnesium stearate or calcium
W094/15620 PCT~S94/00585
2~3~5~ -18-
stearate, and/or polyethylene glycol- Dragee coreq may be
provided with suitable coatinsfi which, if desired, may be
re~istant to gastric juices. For this purpose, concentrated
sugar solutions may be used, which may optionally contain
gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol
and/or titanium dioxide, lacquer solutions and suitable
organic solvents or solvent mixtures. In order to produce
coatings resistant to gastric juices, solution~ of suitable
cellulose preparations such as acetylcellulose phthalate or
hydroxypropylmethylcellulose phthalate, are used. Dyestuffs
and pigments may be added to the tablets of dragee coatings,
for example, for identification or in order to characterize
different combination~ of active compound do~e~.
Other pharmaceutical preparation~ which can be used
orally include push-fit cap~ule~ made of gelatin, as well as
soft, ~ealed cap~ules made of gelatin and a plasticizer such
as glycerol or sorbitol. The push-fit capsules can contaln
the oligonucleotides in the form of granule~ which may be
mixed with filler~ such as lactose, binders such as
starche~, and/or lubricants such ac talc or magne~ium
stearate and, optionally, ~tabilizer~. In soft capsules,
the active compoundq are preferably dissolved or suspended
in suitable liguids, such as fatty oils, liquld paraffin, or
liquid polyethylene glycolQ. In addition, stabilizer~ may
be added.
Po~s$ble pharmaceutical preparations which can be used
rectally include, for example, suppo~itories, which consis~
of a combination of the active compounds with a suppository
base. Suitable ~uppo~itory ~ase~ are, for example, natural
or synthetic tr~glycerides, paraffin hydrocarbons,
polyethylene glycols, or higher alkanols. In addition, i t
is also possible to u~e gelatin rectal capsules which
consist of a combination of the active compounds with a
ba~e. Possible ba~e materials include, for example, liqu~d
~094/15620 ~S 7 PCT~S94/00585
--19-- -
triglycerides, polyethylene glycols, or paraffin
hydrocarbon~.
Sultable formulations for parenteral administration
include aqueous ~olutions of the active compounds in
water-soluble or water-di~persible form. In addition,
suspension~ of the active compounds as appropriate oil
injection suspensions may be admini~tered. Suitable
lipophilic solvent~ or vehicle2 include fatty oil~, for
ex~mple, sename oil, or synthetic fatty acid esters, for
example, ethyl oleate or triglyceride~. Aqueous injection
suspensions may contain sub~tance~ which increase the
viscosity of the ~uspen~ion including, for example, ~odium
carboxymethyl celluloJe, ~orbitol and/or dextran
Optionally, the su~pension may also contain stabilizers.
Additionally, the compound~ of the preacnt invention
may also be admini~tered encapsulated in llpo~omes, wherein
the active inqredient is contained either disper~ed or
variourly preoent in corpuscle~ conci~ting of aqueou~
concentric layera adherent to lipidic layer~. The active
ingredient, depending upon its solubility, may be presen~
both in the aqueous layer, in the lipidic layer, or in what
is generally termed a liposomic ~u~pencion. The hydrophobic
layer, generally but not exclu~ively, compri~es
phospholipid~ ~uch a~ lecithin and ~phingomycelin, steroids
such a~ chole~terol, surfactants such a~ dicetylphosphate,
~tearylamlne, or phosphatidic acid, and/or other materials
of a hydrophobic nature. The diameter~ of the liposomes
generally range from about 15 nm to about 5 microns.
A variety of functional groups, such a~ -OH,-NH2,
-COOH, or -SH, can be attached to the bridging moieties
through linker arms and u~ed to attach con~ugate molecules
which might confer favorable properties to the adduct.
Examples of favorable properties include increased uptake
into the cell, increased lipophilicity or improved binding
WO94/15620 PCT~S94/00585
~1~3~ 20-
to cell surface receptorE. Examples of 8uch conjugate
groupc include, but are not limited to, biotin, folic acid,
chole~terol, epidermal growth factor, and acridine.
The oligonucleotide~ may be used as a diagno~tic probe
Hapten~, such as, but not limited to, 2, 4-dinitrophenyl
group~; vitamins such as biotin and iminobiotin;
streptavidin; fluore~cent moieties such as fluorescein and
FITC; or enzymes such a~ alkaline pho~phata~e, acid
phosphatace, or hor~eradiJh peroxidase, may be attached to
the oligonucleotides. Other labels include, but are not
limited to, detectable marker~ such a~ radioactive nuclides;
and chemical markers including, but not limited to,
biotinated moletie~, antigens, sugar~, fluors, and
pho~phor~, apoenzymes and co-factors, ligands, allosteric
effectors, ferritin, dyes, and micro~phere~. The~e label~
can be attached to any portion of the oligonucleotide which
is not e~ential for binding to its target. Preferably, the
marker i~ attac~ed to the bridging group~. In general, the
bridging group has no biological function, and therefore,
attachment of the label to the bridging group does not
interfere with the therapeutic or diagnostic applications of
the oligonucleotides.
The invention will now be de~cribed with re~pect to the
following e~umple~, the scope of which doe~ not limit the
invention. In particular, the sequences of the paired
oligonucleotidee in the examples hereinafter deccribed
compr~ee a DNA binding ~equence of the tumor ~uppre~sor
protein, p53. Thi~ protein, which i~ mutated in a number of
human cancer~, wae identified a~ a ~equence-~pecific
DNA-binding-protein by Kern, et al., Science, Vol. 252, pgs
1708-1711 (1991). Thc ~ubject of p53 mutations in human
cancers ha~ al~o bee reviewed in Hollstein, et al., Sclence
Vol. 253, pg~. 49-53 (199O).
W094/15620 -21-` 5
ExamPle 1
SYnthesis of an open chain oliqonucleotide with two
hexaethYlene qlYcol bridqing qroups.
An oligonucleotide with the following structure:
5'-DMTr-AGCATGCCXG~CATGCTCAGACATGCCXGGCA~ , whereln A
i8 adenine, C is cytosine, G i8 guanine, T is thymine, X
i-Q hexaethylene glycol phosphodiester, Y is phosphate, and
DMTr is dimethoxytrityl, was synthesized using a DNA
synthesizer.
Synthesis was carried out on a l umole scale using
conventional cyanoethyl pho~phoramidities and other reagents
as a~ follows: The 3'-phosphate was intraduced using
(2-cyanoethoxy)-2-(2'-0-4,4'-dimethoxytritylo~yethyl-
sulfonyl) ethoxy-N, N-diisopropylamino-phosphine (Horn and
Urdea, Tetrahedron Letters, Vol. 27 pgs. 4705-4708 (19~6))
as the phosphoramidite, the reagent being coupled directly
to controlled-pore gla88 ~olid support to which a
deoxycytidine re~idue was attached (i.e., a C-column). The
hexaethylene glycol bridging groups were introduced u-~ing
4,4'-dimethoxytrityloxy-hexaethyleneoxy-2-cyanoethoxy-N,N'-
diisopropylaminopho~phine (Durand et al, Nucleic Acids
Re~earch, Vol. 18, pgs. 63S3-6359 (l990)). After cleavage
from the solid zupport, the agueous ammonia solution wa~
heated at 55C to remove protecting groups and ammonia was
removed by pa-~ing a stream of nitrogen over the solution.
The Jolution wa~ then lyophilized and dissolved in 0.02 M
triethylummonium bicar~onate, pH 7.6. The crude trityl-on
oligonucleotide wa~ purified by reversed phase HPLC (C4
Radial ~ak cartridge, 25 X lO0 mm, 15u, 300A) using a linear
gradient of O.l M triethylammonium acetate
(TEAA)/acetonitrile, with the concentration of acetonitrile
being varied from 2 to 20% over 55 minutes. The peak
eluting between 43 and 50 minutes, corresonding to the
tritylated oligonucleotide, was collected and lyophilized to
W094t15620 PCT~S94/00585
2153~ 22-
remove buffer and detritylated by treatment with O.lM acetlc
acid ~olution for 10 minutes at room temperature. The
product was directly extracted with ethyl acetate (3x)
followed by ether (6x) and lyophilized to dryne~s. The
residue wa~ converted into the sodium salt by dissolution in
water (1 mL) and pa~age through a column of ion exchange
resin (Dowex AGSOW-X8, 7 x lSO mm). The eluate was
evaporated to dryness to give an open chain oligonucleotide
having the following structural formula:
~G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C
C-C-G-T-A-C-G-A G-T-C-T-G-T-A-C-G-G
5' HO po32 3~
wherein X i~ O(C~2 CH20)6 -PO 3-
The above sequence corresponds to a portion of the DNA~equence which i~ known to bind to the p53 protein encoded
by the p53 tumor suppre~sor gene.
Example 2
Formation of a clo~ed circular oliqonucleotide with two
hexaethYlene qlycol bridqing groups.
The oligonucleotide isolated from Example 1 (10 OD260
units) was dlaaolved in sodium 4-morpholine-ethanesulfonate
buffer (MES, 0.05 M,pH 6.0, 22 uL) containing 20 mM
magneaium chlorlde and treated with
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) (EDC, 6.4
mg). The mixture wa~ briefly vortexed, ~tored at 4C and
analyzed by RPLC u~ing a Dionex PA-100 column, 4 x 250 mm.
(Buffer A:25 mM tri~-chloride, pH 8, containing 0.5X
acetonitrile; buffer B: 25mM tris-chloride, lM ammonium
chloride, pH 8, containing 0.5~ acetonltrile. Flow rate 1.5
mL/min, gradient: 15X B to 70/0 B over 5 min., then 70-80~
5-20 min.) Since analysis after 15 hour~ indicated that a
~094/15620 PCT~S94/00585
~;g
substantial amount of ~tarting material remained, the
oligonucleotide wa8 precipitated by addition of absolute
ethanol (85 uL), redissolved in MES buffer (22 uL) and
treated with additional EDC (~3.7 mg). After storage for 2
days at 4C, the ethanol precipitation procedure was
repeated and the oligonucleotide was treated for a third
time with EDC (5.3 mg) in MES (22uL) for 24 hours at 4C.
HPLC analy~is at this point indicated that no ~tarting
material remained. The oligonucleotide was precipitated by
addition of ethanol (B5 uL), (washed with 300 uL absolute
ethanol), and dried to give a clo~ed, circular,
oligonucleotide having the following structural formula:
~ G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C ~
X X
~C-C-G-T-A-C-G-A-G-T-C-T-G-T-A-C-G-G~
wherein X is O(CH2CH20)6 3
Analy~i~ by polyacrylamide gel electrophoresis (15%
acrylamide, 7M urea, tris borate/EDTA buffer), followed by
W detection, revealed a ~ingle, fa~ter running band as
compared to the unligated material.
Example 3
Synthesis of an Open Chain Double-Stranded Oliqonucleotide
with Two Dodecanediol ~ridqinq Group~
a) Synthe~i~ of 4,4'-dimethoxytrityldodecanediol-2-
cyanoethoxy-N,N-di-i~opropylamino-phosphine.
Dodecanedlol (1.012 g, 5 mmol) wa~ dried by
coc~poration wlth di~tilled pyridine (2 x 10 mL), dissolved
in distilled pyridine (20 mL) under nitrogen, and while
being stirred wa~ treated with dimethoxytrityl chloride
(1.694 g, 5 mm~1). The reaction was monitored by TLC uslng
methanol/methylene chloride (1:9) as the solvent system and
after 3.5 hour~ at room temperature the mi~ture wa~
partitioned between methylene chloride (100 mL) and 5%
W094/15620 PCT~S94/00585
~1S3~7
-24-
aqueou~ sodium bicarbonate (80 mL)- The organic layer was
wa~hed with 5% sodium bicarbonate (2 x 80 mL) followed by
~aturated sodium chloride (80 mL) and concentrated to a gum
The sample wa~ purified by column chromatography on silica
gel (80 g, 230-400 mesh) u~ing a linear gradient of methanol
in methylene chloride/triethylamine (99.8:0.2). The
concentration of methanol was rai~ed in a stepwise manner
from 0.5-7~. The appropriate fractions were combined and
evaporated to yield 1.16 g (2.30 mmol, 46%) of
mono-(4,4'-dimethoxytrityl)-dodecanediol as a yellowish gum.
A sample of mono-(4,4'-dimethoxytrityl)-dodecanediol
(1.16 g, 2.30 mmol) was dis~olved in dimethylethylamine
(1.24 mL, 5x) and methylene chloride (15 mL) under nitrogen
and, while being stirred wa~ treated with 2-cyanoethyl N,
N-dii~opropylamino-chloropho~phine (1 g, 4.225 mmol, 1.8 x).
After 2.5 hr~. at room temperature, the reaction was checked
by TLC u~ing ethyl aeetate/triethylamine (95:5) as the
solvent system, and ~ince the reaction was incomplete,
additional 2-cyanoethyl-N, N-dii~opropylamino-
chloropho~phine (lg, 4.225 mmol, 1.8 x) was added. After an
additional 0.5 hr., TLC ~howed the reaction to be
~ub~tantially complete. The mixture wa~ partitioned between
ethyl acetate (80 mL) and 5% sodium bicarbonate (100 mL) and
the organic layer was washed with 5% ~odium bicarbonate (2 x
100 mL) followed by ~aturated ~odium chloride (100 mL) and
concentrated to gum. The sample was purified by column
chromatoqraphy on ~ilica gel (50 g, 230-400 me~h) using
ethyl acetate/triethylamine (99.8:0.2). The appropriate
fraction~ were combined and evaporated to yield 1.454 g
(2.06 mmol, 89.6X) of dimethoxytrityldodecanediol-
2-cyanoethoxy-N,N-di-isopropylamino-pho~phine as a yellowish
gum.
V094/15620 ~ S PCT~S94/00585
~ 3~S~
b) Oligonucleotide synthesis
An oligodeoxynucleotide with the following ~tructure ~s
synthesized using a DNA ~ynthesizer:
5'-DMTr-AGCATGCCTXAGGCATGCTCAGACAlG~lXAGGCAl~l~l~Y, where
A = adenine, C = cyto~ine, G = guanine, T = thymine, X =
dodecanediol-phosphodiester brid~ing group, Y = phosphate
and DMTr = dimethoxytrityl.
Synthesis is carried out on a 1 umole scale using
conventional cyanoethyl phosphoramidites and other reagents
as follows: The 3' phosphate is introduced as described in
Example 1, the reagent being coupled directly to controlled-
pore glass solid support to which a deoxycytidine residue
was attached (i.e. a C-column). The dodecanediol-
pho~phodiester bridging groups are introduced using
4,4'-dimethoxytrityloxy-dodecanediol-2-cyanoethoxy-N,N'-
diisopropylamino-pho~phine. After cleavage from the solid
support, the aqueous ammonia solution is heated at 55C to
remove protecting groups and ammonia is removed by passing a
stream of nitrogen over the solution. The solution is then
lyophilized and di~solved in 0.02 M triethylammonium
bicarbonate, pH 7.6. The crude, trityl-on oligonucleotide
is purified by rever~ed phase HPLC (C4 Radial Pak cartridge,
25 x 100 mm, 15u, 300A) u~ing a linear gradient of 0.1 M
triethylammonium acetate (TEM )/acetonitrile, with the
concentration of acetonitrile being varied from 2 to 20 %
over 55 minute~. The peak corresponding to the tritylated
oligonucleotide is collected and lyophilized to remove
buffer and detritylated by treatment with O.lM acetic acid
solution for 10 minutes at room temperature. The product is
directly extracted with ethyl acetate (3x) followed by ether
(6x) and lyophilized to dryness. The re~idue is converted
into the sodium salt by dissolution in water (1 mL) and
passage through a column of ion exchange re~in (Dowex
AGSOW-X8, 7 x 150 mm). The eluate is evaporated to dryness
W0 94/15620 21~ 3 ~ ~ ~ PCT/US94/00~85
-26-
to give the open chain double-stranded oligonucleotide with
two dodecanediol bridging groups. The purity of the product
is examined by reinjection into an analytical ion exchange
Dionex PA-100 column, 4 x 250 mm. Buffer A: 25 mM
tris-chloride, pH 8 containing 0.5% acetonitrile, buffer B:
25 mM tris-chloride, lM sodium chloride, pH 8 containing 0.5
% acetonitrile. Flow rate 1.5 mL/min. Gradient: 15% B to
70% B over 5 min, then 70-80% B 5-20 min. This material of
the following structure is suitable for chemical ligation as
described hereinbelow in Example 4.
~A-G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C-T ~
X X
~T-C-C-G-T-A-C-G-A G-T-C-T-G-T-A-C-G-G-A
5'H0 opo32 3,
where X is 0-(CH2)12-0-PO3
Example 4
Synthesis of a Closed, Circular Double-Stranded
Oliqonucleotide with Two Dodecanediol Brid~inq Groups
The oligonucleotide isolated from Example 3 (10 OD260
units) i~ di~solved in sodium 4-morpholine-ethanesulfonate
buffer (MES, 0.05 M, pH 6.0, lmL) containing 20 mM magnesium
chloride and treated with
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 100 mg)
The mixture i~ briefly vortexed, stored at 4C for 2 days,
and the oligonucleotide is precipitated by addition of
absolute ethanol (9 mL), washed with 1 mL absolute ethanol,
and dried to give the closed circular oligonucleotide.
Analysis by polyacrylamide gel electrophoresis (15%
acrylamide, 7M urea, tris borate/EDTA buffer, W detection),
reveal~ a single, faster running band as compared to the
unligated material. HPLC analysis employed a Dionex PA-100
column, 4 x 250 mm (Buffer A: 25 mM tri~-chloride, pH 8
W094/15620 ~ PCT~S94/~585
s3~ ~-
-27- ~ ~
containing 0.5X acetonitrile: Buffer B: 25 mM
tris-chloride, lM ~odium chloride, pH 8 containing 0.5%
acetonitrile. Flow rate: 1.5 mL/min. Gradient: 15% B to
70% B over 5 min. then 70-80x B 5-20 min). This procedure
provides material having the following structure:
~A-G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C-T \
X X
~T-C-C-G-T-A-C-G-A-G-T-C-T-G-T-A-C-G-G-A~
where X i 8 O- (CH2)12-0-P03
Example 5
Synthe~is of an Open Chain, Double-Stranded Oliqonucleotide
with Biotin Attached to one of the Bridqing Groups
An oligodeoxynucleotide with the following structure is
prepared using a DNA synthesizer:
S'-DMTr-AGCA~ XAGGCATGCTCAGACA~L~AGGCA~ Y, _
where A i~ adenine, C is cytosine, G is guanine, T i 5
thymine, W i~ hexaethylene glycol pho~phodiester, X is
triethylene glycol, Y is phosphate, Z is
2(4-biotinamidopentyl)-1,3-propanediol-phosphodiester, and
DMTr = 4,4'-dimethoxytrityl.
Syntho~is i8 carried out on a 1 umole ~cale using
conventional cyanoethyl phosphoramidites and other reagents
as follows: The 3'- phosphate and the hexaethylene glycol
group are introduced as described in Example 1 and the
triethylene glycol groups are introduced u~ing
4,4'-dimethoxytrityloxy-triethyleneoxy-2-cyanoethoxy-N,N'-
diisopropylaminopho~phine, obtalned from Glen Research
Corporation, Sterling, Virginia. The biotin is introduced
using l-(4,4'-dimethoxytrityl)-2~4-biotinamidopentyl)-1,3-
propanediol-3- (2-cyanoethyl~-N, N-diisopropylamino-
chlorophosphine, al~o obtalned from Glen Research
Corporation.
W094/15620 PCT~S94/00585
21.53 057 28
After cleavage from the solid support, the aqueous
ammonia solution is heated at 55C to remove protecting
groups and ammonia is removed by passing a ~tream of
nitrogen over the ~olution. The solution is then
lyophilized and purified by rever~ed-phase HPLC. The
product is directly extracted with ethyl acetate (3x)
followed by ether (6x) and lyophilized to dryness. The
residue is converted into the sodium salt by dissolution in
water (1 mL) and passage through a column of ion exchange
resin (Dowex AG50W-X8, 7 x 150 mm). The eluate i8
evaporated to dryness to give the open chain double stranded
oligonucleotide having the following ~tructure, with biotln
in one of the bridging groups.
~ X
Z A-G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C-T
W
~X -T-C-C-~-T-A-C-G-A G-T-C-T-G-T-A-C-G-G-A ~
l l
5' H0 po32
where W is O(CH2CH20)6 P03
X i~ O(CH2CH20)3 P03
Z i ~ OCH2CHCH20-P03
CH2CH2CH2CH2NH-Biotin
Thi~ material is suitable for chemical ligation as
de~cribed in Example 6.
Example 6
Synthe~is of a Closed, Circular Double-Stranded
Oliqonucleotide with Biotin Attached to one of the Bridqing
Groups
The oligonucleotide isolated from Example 5 (10 OD260
units) is di~olved in sodium 4-morpholine-ethane~ulfonate
WO94/15620 ~S~O PCT~S94/00585
-29-
buffer (MES, 0.05 M, pH 6-0, 22 uL) containing 20 mM
magnesium chloride and treated with
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 6.4 mg)
The mixture is briefly vortexed, stored at 4C for 15 hrs.
and the oligonucleotide is precipitated by addition of
absolute ethanol (85 uL), redissolved in MES buffer (22 uL)
and treated with additional EDC (8.7 mg). After storage for
2 day~ at 4C, the ethanol precipitation procedure is
repeated and the oligonucleotide is treated for a third tlme
with EDC (5.3 mg) in MES (22uL) for 24 hours at 4C. The
oligonucleotide is precipitated by addition of ethanol (85
uL), washed with 300 uL absolute ethanol, and dried to give
the closed circular oligonucleotide having the following
structure:
~ X~
Z A-G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C-T
\ T-C-C-G-T-A-C-G-A-G-T-C-T-G-T-A-C-G-G-A
~X~
where W is o(CH2cH2o)6 PO3
X is OlCH2CH2O)3-PO3
Z is OCH2CHCH2O-PO3
\
CH2CH2CH2CH2NH-Biotin
Example 7
SYnthe~is of an oliqonucleotlde with peptide bridqing
qroup~.
a) Synthe~iq of a peptide phosphoramidite.
The tripeptide Ala-Ala-Ala (4 mmol) is treated with the
N-hydroxysuccinimide ester of 3-hydroxybutyric acid (4 mmol)
in dimethylformamide (20 mL) at room temperature for 4
hours. After removal of solvent, the residue is dissolved
in pyridine and treated with 4,4'-dimethoxytrityl chloride
W094/15620 PCT~S94/00585
2l53~7 _30_
(2 mmol) at room temperature for 18 hours. The product is
evaporated to dryness, partitioned between ethyl acetate and
aqueous sodium bicarbonate and the organic layer is washed
with sodium bicarbonate (1 x) followed by water (2 x) and
dried over magne 8 ium sulfate. The solution is filtered,
evaporated to drynes~, and purified by silica column
chromatography uqing methylene chloride/methanol/
triethylamine as the solvent to give the dimethoxytritylated
trlpeptide of the following structure:
DMTrO-CH(CH3)CH2-CO-NHCH(CH3)CO-NHCH(CH3)CO-NHCH(CH3)COO~
This material ~2 mmol) is dissolved in dimethylformamide and
treated with 6-aminohexanol (2 mmol) at room temperature for
1~ hours using dicyclohexylcarbodiimide (5 mmol) as the
coupling agent. The urea is removed by filtration and the
product is evaporated to dryness, partitioned between ethyl
acetate and aqueous sodium bicar~onate, and the organic
layer is washed w1th sodium bicarbonate (1 x) followed by
water (2 x) and dried over magnesium sulfate. The solution
is filtered, evaporated to dryness, and purified by silica
column chromatography using methylene
chloride/methanol/triethylamine as the solvent to give the
dimethoxytr1tyl-ted tripeptide aminohexanol derivative of
the following ~tructure:
DMTrO-C~(CH3)CH2-CO-NHCH(CH3)CO-NHCH(CH3)CO-NHCH(CH3)CO-
NH(CR2)6 OH
The aminohexanol derivative (1 mmol) is dissolved in
methylene chloride containinq diisopropylethylamine (2 mmol)
and treated w1th 2-cyanoethyl-N,N-diisopropylamino-
chlorophosphine (2 mmol) at room temperature for 20 min.
The ~olution is poured into ethyl acetate, extracted with 5~0
WO94/15620 ~ S PCT~S94/00585
-31- 5~
aqueou~ sodium bicarbonate (2 x) followed by saturated
aqueous sodium chloride (2 x) and dried over magnesium
chloride overnight. The solid is removed by filtration and
the solution is evaporated to dryness and purified by sillca
column chromatography using methylene
chloride/methanol/triethylamine as the solvent to give the
phosphoramidite with the following structure:
DMTrO-CH(CH3)CH2CO-NHCH(CH3)CO-NHCH(CH3)CO-NHCH(CH3)CO-
( H2)6 O-p(ocH2cH2cN)N(ipr)2
This material is u~ed to attach the bridging groups to the
oligonucleotide in the DNA synthesizer.
b) Oliqonucleotide synthesis
The procedures outlined in Examples 1 and 2 are
followed to synthesize oligonucleotides with peptide
bridging groups, except that the peptide pho~phoramidite of
the present example i~ ~ubstituted for the bridging group
phosphoramidite described in Example 1.
This procedure can also be used to attach polyamides as
bridging groups for oligonucleotides.
Example 8
Synthesis of an oliqonucleotide with polYamine bridqinq
qroups
The polyamine spermidine (4 mmol) is treated with
~ -butyrolactone to give the disubstituted derivative of
the following structure:
HO(CH2)3CO-NH(CH2)3NH(CH2)4NH-CO(CH2)3-OH
This material is dissolved in pyridine (lO mL) and
treated with trifluoroacetic anhydride (2 mL) overnight at
room temperature. The solution is treated with water at 0C
WO94/15620 2 15 3 0 5 ~ - 32- PCT~S94/00585
for 4 hours and evaporated to dryness to give the
N-trifluoroacetyl derivative which is treated with
4,4-dimethoxytrityl chloride (4 mmol) for 19 hours at room
temperature and evaporated to dryness. The residue l5
partitioned between ethyl acetate and aqueous sodium
bicarbonate and the organic layer is washed with water (2 x)
and dried over magnesium sulfate overnight. The solld i 5
removed by filtration and the solution is evaporated to
dryness and pur~fied by silica column chromatography.
FractionQ containing the monotrityl derivative are combined,
evaporated to dryness and 1 mmol of this material is
di~solved in methylene chloride containing
diisopropylethylamine (2 mmol) and treated with
2-cyanoethyl-N,N-diisopropylamino-chlorophosphine (2 mmol)
at room temperature for 20 min. The ~olution is poured into
ethyl acetate, extracted with 5~ aqueou~ sodium bicarbonate
(2 x) followed by saturated aqueou~ ~odium chloride (2 x)
and dried over magnesium chloride overnight. The aolid is
removed by filtration and the solution i8 evaporated to
dryne~s and purified by silica column chromatography u~ing
methylene chloride/methanol as the ~olvent to give the
phosphoramidite with the following structure:
DMTr-O(CH2)3CO-NH(CH2)3~H(CH2)4NH CO(CH2)3 2 2
~OCF3 N (iPr)2
This material i~ used in the DNA synthesizer to introduce
bridging groups into the oligonucleotide. The procedures
outlines in Examples 1 and 2 are followed to synthesize
oligonucleotides with polyamine bridging groups, except that
the bridginq group phosphoramidite of the present example is
substituted for the bridgin~ group phosphoramidite deqcribed
in Example 1.
W094/15620 2~ PCT~S94/00585
s30~
Example 9
IncorPoration of PolYalkylene thioqlYcol bridqinq qroups
3,6-Dithio-1,8-octanediol is treated with
4,4-dimethoxytrityl chloride in pyridine and then converted
into a phosphoramidite derivative of the following structure
by reaction with 2-cyanoethyl-N,N-diisopropylamino-
chlorophosphine using the procedure described in Example 3:
H2cH2scH2cH2scH2cH2O-P-N(ipr)
OCH2CH2CN
This material is employed as the bridging group
phosphoramidite in the synthesi~ of an oligonucleotide as
described in Example 1.
Example lO
Synthesis of Double-Stranded Oligonucleotides with Two
Di~ub~tituted Aromatic ~rid~inq Groups
An oligodeoxynucleotide with the following structure 15
~ynthesized-uJing a DNA synthesizer:
5'-DMTr-AGCA-~L~AGGCATGCTCAGACA~ ~AGGCA~
wherein A = adenine, C = cytosine, G = guanine, T = thymlne
X =0-(CH2)6-NHCO-C6H5-CONH-(CH2)6-OP03 bridging group,
Y = pho~phate and DMTr = dimethoxytrityl.
Synthesis i8 carried out on a 1 umol scale using
conventional cyanoethyl pho3phoramidites and other reagent~
as follows: The 3' phosphate is introduced a~ de~cribed i~
~xample 1, and the aromatic bridging groups are introduced
u~ing N-(6-(4,4'-dimethoxytrityloxy)hexyl)-N'
-(6(2-cyanoethoxy-N,N'- diisopropylamino-
WO94/15620 PCT~S94/00585
2 lS 3 ~ _34_
pho~phinyloxy)hexYl)terePhthalamide as de~cribed by Cashmanet al in the Journal of the American Chemical Society, Vol
114, pqs 8772-8777 (1992) After cleavage from the solid
support, the oligonucleotide i9 proce~ed as described ln
Example 1 to give an open chain oligonucleotide duplex with
two aromatic bridging groups Formation of the closed
circular duplex i~ carried out using the procedure outlined
in Example 2
Example 11
Incorporation of an intercalating aqent as a bridqing ~roup
The intercalating agent acriflavine is treated with
6-bromo-1-hexanol to form a disubstituted derivative which
is then treated with one equivalent of 4,4-dimethoxytrityl
chloride to give the monotrityl compound having the
following structure
~r~O~~Cff~ ,fLk_o~
After purification and i~olation by ~ilica column
chromatography, the monotrityl compound i5 treated with
trifluoroacetic nhydride in pyridine followed by aqueous
workup to ~ive the N-trifluoroacetyl derivative which is
then converted into a phosphoramidite of the following
structure by tre-tment with 2-cyanoethyl-N,N-
diisopropylamino-chlorop ~ s described in Example 3
D~T~-o~ ) ocf~zChCl/
WO94/15620 ~ PCT~S94100585
-35- ~
The phosphoramidite is incorporated into an oligonucleotide
by the procedure outlined in Example 1.
ExamPle 12
IncorPoration of a carbohydrate bridqinq moiety
6-O-~-D glucopyranosyl-D-qlucopyranose (B-gentobiose)
is treated w~th t-butyl-dimethylsilyl chloride to produce
the 6'-silyl compound which is converted into the acetobromo
derivative by a conventional method using acetic anhydride
followed by hydrogen bromide in acetic acid. The l-bromo
derivative i~ then treated with 1,6- hexanediol to give the
glycoside which i9 reacted with 4,4- dimethoxytrityl
chloride to give a compound having the following structure:
~4to~ 7~0~,--~C~f2)C_o~
The 8ilyl group i~ ~c...ovc~ u~ing fluoride ion and the
6-hydroxy compound is treated with 2-cyanoethoxy-N,N-
dii opropylamino-chlorophosphine to give a phosphoramidite
having the following ~tructure:
C~f~z C~ O~
tir~ ~ ~ ~ ~ t~ )6- ~ ~
~C ~o
~C
W094/15620 2 1 5 3 5~ PCT~S94/00585
-36-
This material is employed in the DNA synthesizer to
introduce bridging groups as described in previous examples.
Example 13
Thermal Denaturation of Bridqed, Double-Stranded
OliqonucLeotides
The thermal denaturation temperatures (Tm's) of some of
the oligonucleotide~ of the present invention were measured
on a Gilford spectrometer at 260 nm in order to determine
their relative ~tabilitie fi . Approximately 1 OD260 unit of
each oligonucleotide was dis~olved in O.9 mL of 10 mM
disodium pho~phate buffer, pH 7.0, and each sample was
heated briefly at 100C, and allowed to cool 910wly to room
temperature. Melting profiles were obtained by increasing
the temperature of the ~ample~ from 25C to 100C at a rate
of 0.8C per minute, followed by measurement of optical
absorption at each time interval. The oligonucleotides
examined in thie study are as follows:
A-G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C-T X
\T-C-C-G-T-A-C-G-A G-T-C-T-G-T-A-C-G-G-A
5' HO ~po32 3~
Oligonucleotide 1. X = pentathymidylate (T5)
Oligonucleotide 2. X = triethylene glycol phosphodiester
~ -G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C-T
X X
~T-C-C-G-T-A-C-G-A-G-T-C-T-G-T-A-C-G-G-A
Oligonucleotide 3. X = T5
Oligonucleotide 4. X = triethyiene glycol phosphodiester
WO94/15620 . ~ PCT~S94/00585
A-G-G-C-A-T-G-C-T-C-~-G-A-C-A-T-G-C-C-T
Oligonucleotide 5.
T-C-C-G-T-A-C-G-A-G-T-C-T-G-T-A-C-G-G-A
Oligonucleotide 6.
The thermal denaturation temperature~ of
OligonucleotideR l through 6 are given in Table I below.
Table I
Oligo- 3'-Terminal Bridging Tm
nucleotide Group GrouP (X~ ~C)
1 Pho~phomono- T5 61.5
ester
2 Phosphomono- Triethylene 68
e~ter glycol
pho~phodie~ter
3 None T~ 82.5
4 None Triethylene 89.5
glycol
pho~phodie~ter
5 and 6 N/A None 62
Thi~ e~periment demonstrates that an open chain,
double-~tranded ollgonucleotide with triethylene glycol
bridging group~ i~ more stable towardq thermal denaturation
than either the ~ame ~equence with pentathymidylate bridgin~
group~, or an unmodified duplex without any bridging groups.
The clo~ed, circular double-stranded oligonucleotide with
triethylene glycol bridging groups iq also more stable than
the same ~equence with pentathyamidylate bridge~.
W094/15620 1 PCT~S94/00585
30~
-38-
Example 14
EnzYmatic Stability of a Double-Stranded Oliqonucleotide
with Hexaethylene Glycol Bridqing Groups
Enzymes:
Exonuclease - Exonuclea~e III
Endonuclease - Mung Bean Nuclea~e
Buffers:
For Exonuclease III: 50 mM Tri~-HCl, pH 7.5; 5mM MgC12;
5 mM DTT; 50 mg/mL BSA.
For Mung Bean Nuclease. 30 mM NaOAc, pH 5.0; 50 mM NaCl;
1 mM ZnC12; S~ glycerol.
The enzymatic degradation of the detritylated
oligonucleotide of Example 1, having hexaethylene glycol
bridging groups, was compared to a duplex of the same
sequence without any bridging groups. A 1 OD260 sample of
each oligonucleotide was digested in a mixture of 95 ~L of
the reaction buffer and 5 ~L of the enzyme solution
containing 1 unit of the enzyme. The mixture~ were
incubated at 37C (for Exonuclease III) or room temperature
(for Mung Bean Nuclea~e) and the extent of degradation was
monitored by ~PLC wtth a Dionex ion-exchange column, uslng a
linear gradient of ammonium chloride (O-lM) in tris
hydrochloride. The time required for ~O% degradation (tl/2)
for each sample was determined and the following results
were obtained:
WO94/15620 Sob; PCT~S94/00585
-39-
Oliqonucleotide Mung Bean Nuclease Exonucleaqe II'
(tl/2) (t~/2)
Detritylated 68 hours > 80 hours
Oligonucleotide of
Example l
Unmodified 3 hour~ 2.5 hours
duplex
This experiment demonstrate~ that the oligonucleotide
of Example l, posse~sing hexaethylene gly~ol bridging
groups, i8 considerably more re~istant to degradation than
an unmodified duplex of the same sequence.
Example 15
Bindinq of an Open Chain Double-stranded Oliqonucleotide
with Two Hexaethylene Glycol Bridqinq Groups to pS3 Tumor
Supprersor Gene Protein
The following oligodeoxynucleotider were prepared for
bind to pS3 protein.
l. G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C
~ C-C-G-T-A-C-G-A G-T-C-T-G-T-A-C-G-G
Il
5' HO OH 3'
where X O(CH2CH2)6 P 3
2. G-G-C-A-T-G-C-T-C-A-G-A-C-A-T-G-C-C
3. C-C-G-T-A-C-G-A-G-T-C-T-G-T-A-C-G-G
W094/15620 21~ 3 a 5 ~ PCT~S94/00585
-40-
Oligonucleotide 1 was prepared by the procedure
outlined in Example 1, except that the 3'-phosphoryla~ion
reagent was omitted and the hexaethylene glycol bridging
groups were introduced using
4,4'-dimethoxytrityloxy-hexaethyleneoxy-2-cyanoethoxy-N,N'-
diisopropylaminophosphine as the bridging group reagent. A
92 base pair natural duplex containing a randomized internal
60 base pair region wa~ used as the control oligonucleotide.
The oligonucleotides were radiolabeled with 32p using a
standard protocol as de~cribed in "Molecular Cloning, a
Laboratory Manual" by Sambrook, FritRch and Maniatis, page
11.31, Cold Spring Harbor Pre~ (1989). The radiolabeled
oligonucleotides were then purified and u~ed in an
immunoprecipitation assay to evaluate binding efficiency to
p53 tumor suppressor gene protein. The immunoprecipitation
assay was performed with 2.0 pmole~ purified p53, 0.25
pmoles radiolabeled oligonucleotide, 100ng poly dl-dC, and
400ng each of anti-p53 antibodie~ pAb421 and pAbl801
(purchased from Oncogene Science~, Inc.), incubated in 100
~1 of binding buffer containing 100 mM NaCl, 20 mM Tris pH
7.2, 10% glycerol, 1~ NP40, and 5 mM EDTA a 4C for 1 hour.
The DNA-pS3-anti-pS3 antibody complexes were precipitated
following the addition of 30 ~1 of a 50X slurry of protein A
~epharo~e and mixing at 4C for 30 minutes. After removal
of tho aupernatant, the immunoprecipitate was waRhed three
times with binding buffer. ~ound oligonucleotide was then
quantified by direct Cerenkov counting. Specific binding
wa~ evaluated by comparl~on to an immunoprecipitation
performed in the ab~ence of pS3. The results are summarlzed
below.
WO94/15620 PCT~S94/0058~
-41- ~S30~7
p53 Bindinq of natural and modified oliqonucleotide duplexes
Oliqonucleotide Percent Bound
(vs unmodified duplex~
2 + 3 lOO
l 43.5
control 3.2
This experiment shows that the open chain,
double-~tranded Oligonucleotide l, with he~aethylene glycol
bridging groups, i~ capable of binding to p53 protein
although somewhat le~ efficiently than an unmodified duplex
of the same sequence. This result, taken together with the
results of Ex~mple 14 (which demonstrated ~at an open chain
double-~tranded oligonucleotide with hexaethylene glycol
bridging group~ was considerably more stable towards
nucleases than an unmodified duplex), indicates that an
oligonucleotide with bridging group~ of this type has
considerably greater pharmacological potential.
Advantage~ of the present invention include increased
resistance of the circular oligonucleotide~ to enzymes which
degrade ol~gonucleotide~ by attack at the 5' and/or 3'
termini, ~uch a~, for example, 3' exonuclea~es. In
addition, double-~tranded oligonucleotide~ of the present
invention are re~iJtant to enzymes which degrade
single-~tranded regions of DNA because the non-nucleotide
bridging group~ cannot be recognized by such enzymes. The
bridging group~ can be constructed from simple, readily
available starting materials, and may be lncorporated easlly
into an oligonucleotide using a DNA synthe~izer. In
addition,.both the open chain and the clo~ed, circular,
W094/15620 .~ PCT~S94/00585
2l53~5 l
-42-
paired oligonucleotides with non-nucleotide bridging groups
are capable of forming more stable hydrogen-bonded
structures than the corresponding sequences with nucleotlde
(pentathymidylate) bridging groups or with natural duplexes
without bridging groups and thus provide binding to target
protein~ which are capable of binding double-stranded
oligonucleotides. ~ecause the paired oligonucleotides also
remain hydrogen-bonded at higher temperatures this could be
advantageous for diagnostic applications. Unpaired circular
oligonucleotides might possess a significant advantage over
single-stranded oligonucleotides in binding to
double-~tranded target DNA or RNA by forming Hoog~teen
nteractions with both strands of the target DNA or RNA.
Also, the bridging moieties of the double-stranded
oligonucleotides may be modified to introduce favorable
properties into the molecules, such as increaJed
lipophilicity, or be modified to introduce materials which
assist in the delivery of the oligonucleotide into the cell,
such as cationic group~ or molecules which are recognized by
cell surface receptor~.
It is to be understood, however, that the qcope of the
present invention i~ not to be limited to the specific
embodiments described above. The invention may be practiced
other than a~ particularly described and still be within the
scope of the accompanying claims.
