U.S. patent number 6,172,208 [Application Number 07/908,376] was granted by the patent office on 2001-01-09 for oligonucleotides modified with conjugate groups.
This patent grant is currently assigned to Genzyme Corporation. Invention is credited to Alan Frederick Cook.
United States Patent |
6,172,208 |
Cook |
January 9, 2001 |
Oligonucleotides modified with conjugate groups
Abstract
An oligonucleotide wherein at least one nucleotide unit of the
oligonucleotide is conjugated with a moiety selected from the group
consisting of: (a) amino acids; (b) dipeptide mimics; (c) sugars;
(d) sugar phosphates; (e) neurotransmitters; (f)
poly-hydroxypropylmethacrylamide; (g) dextrans; (h) polymaleic
anhydride; (i) cyclodextrins; (j) starches; and (k)
polyethyleneimine. The oligonucleotides may be employed for binding
to an RNA, and DNA, a protein, or a peptide to inhibit or prevent
gene transcription or gene expression, to inhibit or stimulate the
activities of target molecules, or the oligonucleotides may be
employed as diagnostic probes for determining the presence of
specific DNA or RNA sequences or proteins.
Inventors: |
Cook; Alan Frederick (Cedar
Grove, NJ) |
Assignee: |
Genzyme Corporation
(Framingham, MA)
|
Family
ID: |
25425689 |
Appl.
No.: |
07/908,376 |
Filed: |
July 6, 1992 |
Current U.S.
Class: |
536/23.1;
536/24.3; 536/24.5 |
Current CPC
Class: |
C07H
21/00 (20130101); A61K 47/51 (20170801); C12Q
1/6876 (20130101) |
Current International
Class: |
A61K
47/48 (20060101); C12Q 1/68 (20060101); C07H
21/00 (20060101); C07H 021/04 () |
Field of
Search: |
;536/23.1,24.3,24.5
;514/44 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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9106556 |
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May 1991 |
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WO |
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9110671 |
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Jul 1991 |
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WO |
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Other References
Lemaitre et al., Proc. Natl. Acad. Sci. USA 84, 648-652 (1987).*
.
Vestweber & Schatz, Nature 338, 170-172 (1989).* .
Leamon & Low, Proc. Natl. Acad. Sci. USA 88, 5572-5576 (1991).*
.
Hoflack & Kornfeld, J. Biol. Chem. 260(22), 12008-14 (1985).*
.
Uhlmann, et al., Chemical Reviews, vol. 90, No. 4, pp. 544-584
(Jun. 1990)..
|
Primary Examiner: Horlick; Kenneth R.
Attorney, Agent or Firm: Olstein; Elliot M. Lillie; Raymond
J.
Claims
What is claimed is:
1. An oligonucleotide wherein each nucleotide unit of the
oligonucleotide includes a sugar moiety, a purine or pyrimidine
base, and a phosphorus-containing moiety, wherein said
oligonucleotide is conjugated to at least one sugar phosphate
moiety which is independent of a nucleotide unit, wherein said at
least one sugar phosphate moiety is attached at said purine or
pyrimidine base, at said phosphorus-containing moiety, or at said
sugar moiety of said oligonucleotide.
2. A composition for binding to an RNA, a DNA, a protein, or a
peptide comprising:
(A) an oligonucleotide wherein each nucleotide unit of the
oligonucleotide includes a sugar moiety, a purine or pyrimidine
base, and a phosphorus-containing moiety wherein said
oligonucleotide is conjugated to at least one sugar phosphate
moiety which is independent of a nucleotide unit, wherein said at
least one sugar phosphate moiety is attached at said purine or
pyrimidine base, at said phosphorus-containing moiety, or at said
sugar moiety of said nucleotide, and
(B) an acceptable pharmaceutical carrier, wherein said
oligonucleotide is present in an effective binding amount to an
RNA, a DNA, a protein, or a peptide.
3. A probe for determining the presence of a target DAN or RNA
sequence, comprising:
an oligonucleotide wherein each nucleotide unit of the
oligonucleotide includes a sugar moiety, a purine or pyrimidine
base, and a phosphorus-containing moiety, wherein said
oligonucleotide is conjugated to at least one sugar phosphate
moiety which is independent of a nucleotide unit, wherein said at
least one sugar phosphate moiety is attached at said purine or
pyrimidine base, at said phosphorus-containing moiety, or at said
sugar moiety of said oligonucleotide.
4. The oligonucleotide of claim 1 wherein said sugar phosphate is
selected from the group consisting of mannose-6-phosphate,
glucose-6-phosphate, galactose-6-phosphate, mannose-1-phosphate,
glucose-1-phosphate, galactose-1-phosphate,
6-O-phosphoryl-.alpha.-D-mannopyranosyl-(1-2)-D-mannopyranose,
6-O-phosphoryl-.alpha.-D-mannopyranosyl-(1-3)-D-mannopyranose,
6-O-phosphoryl-.alpha.-D-mannopyranosyl-(1-6)-D-mannopyranose,
6-O-phosphoryl-.alpha.-D-mannopyranosyl-(1-2)-D-mannopyranose-(1-2)-D-mann
opyranose, and pentamannose-6-phosphate.
5. The oligonucleotide of claim 2 wherein said sugar phosphate is
selected from the group consisting of mannose-6-phosphate,
glucose-6-phosphate, galactose6-phosphate, mannose-1-phosphate,
glucose-1-phosphate, galactose-1-phosphate,
6-O-phosphoryl-.alpha.-D-mannopyranosyl-(1-2)-D-mannopyranose,
6-O-phosphoryl-.alpha.-D-mannopyranosyl-(1-3)-D-mannopyranose,
6-O-phosphoryl-.alpha.-D-mannopyranosyl-(1-6)-D-mannopyranose,
6-O-phosphoryl-.alpha.-D-mannopyranosyl-(1-2)-D-mannopyranose-(1-2)-D-mann
opyranose, and pentamannose-6-phosphate.
Description
This invention relates to oligonucleotides which may bind to a DNA,
and RNA, a protein, or a polypeptide, for use as a therapeutic
agent or as a diagnostic probe. More particularly, this invention
relates to oligonucleotides wherein at least one nucleotide unit of
the oligonucleotide includes a conjugate moiety.
Oligonucleotides may be of value as therapeutic agents for the
treatment of a wide variety of diseases. They offer the potential
for a high degree of specificity by virtue of their capability for
interaction with target macromolecules. Natural oligonucleotides,
however, are relatively ineffective as therapeutic agents due to
their poor penetrability into the cell, and their rapid degradation
by enzymes. Therefore, relatively high concentrations of natural
oligos are needed in order to achieve a therapeutic effect.
Applicants have found that the attachment of specific classes of
conjugate groups to oligonucleotides improves their uptake into the
cell, improves their stability, or both. Conjugate groups employed
in the present invention, which include amino acids, dipeptide
mimics, sugars, sugar phosphates, and neurotransmitters or
analogues thereof, have been shown to be transported into the cell
by specific transporter systems. These transporters can be used to
improve the uptake of oligonucleotide conjugates into the cell,
and, depending upon the site of attachment, can also prevent or
reduce degradation.
Hydrophilic polymer conjugate groups which may be employed in the
present invention, and which include
poly-hydroxypropylmethacrylamide, dextrans, polymaleic anhydride,
cyclodextrins, starches, and polyethyleneimine, may be used to
reduce or prevent degradation of the oligonucleotide by blocking
access of the conjugate to the enzymes which degrade
oligonucleotides.
The scientific literature contains descriptions of certain
conjugate groups attached to oligonucleotides.
Conjugates with polylysine have been described by Lemaitre, et al.,
Proc. Natl. Acad. Sci. USA, Vol. 84, pgs. 648-562 (1987), and shown
to be more active in cell culture than their unmodified
counterparts. Polylysine, however, is not a preferred molecule for
conjugation due to its relatively high toxicity. Amphiphilic
oligonucleotide conjugates with polyethylene glycol have been
described by Tullis in U.S. Pat. No. 4,904,582, and conjugates with
cholesterol have been reported by Letsinger et al., (Abstracts,
Conference on Nucleic Acid Therapeutics, Clearwater, Fl., (1991)).
Many more conjugates have been synthesized for diagnostic
applications. In these cases the molecule conjugated to the
oligonucleotide acts as a reporter or signaling group, such as, for
example, oligonucleotides attached to fluorescent groups which
enable the duplex to be detached visually, biotin conjugates which
can be detected after capture by streptavidin attached to a
signaling group, or enzyme conjugates which can directly generate a
signal upon addition of a suitable substrate. Several reviews on
modified oligonucleotides, including conjugates have been
published; see for example, Uhlmann and Peyman, Chemical Reviews,
Vol. 90, pgs. 543-584 (1990), and Goodchild, Bioconjugate
Chemistry, Vol. 1, pgs 165-187 (1990).
In accordance with an aspect of the present invention, there is
provided an oligonucleotide wherein at least one nucleotide unit of
the oligonucleotide is conjugated with a moiety selected from the
group consisting of (a) amino acids; (b) dipeptide mimics; (c)
sugars; (d) sugar phosphates; (e) neurotransmitters; (f)
poly-hydroxypropylmethacrylamide; (g) dextrans; (h) polymaleic
anhydride; (i) cyclodextrins; (j) starches; and (k)
polyethyleneimine.
The term "oligonucleotide" as used herein means that the
oligonucleotide may be a ribonucleotide, deoxyribonucleotide, or a
mixed ribonucleotide/deoxyribo nucleotide; i.e., the
oligonucleotide may include ribose or deoxyribose sugars or a
mixture of both. Alternatively, the oligonucleotide may include
other 5-carbon or 6-carbon sugars, such as, for example, arabiose,
xylose, glucose, galactose, or deoxy derivative thereof or any
mixture of sugars.
The phosphorus-containing moieties of the oligonucleotides of the
present invention may be modified or unmodified. The
pohosphorus-containing moiety may be, for example, a phosphate,
phosphonate, alkylphosphonate, aminoalkyl phosphonate,
thiophosphonate, phosphoramidate, phosphorodiamidate,
phosphorothioate, phosphorothionate, phosphorothiolate,
phosphoramidothiolate, and phosphorimidate. It is to be understood,
however, that the scope of the present invention is not to be
limited to any specific phosphorus moiety or moieties. Also, the
phosphorus moiety may be modified with a cationic, anionic, or
sqitterionic moiety. The oligonucleotides may also contain backbone
linkages which do not contain phosphorus, such as carbonates,
carboxymehtyl esters, acetamidates, carbamates, acetals, and the
like.
The oligonucleotides also include any natural or unnatural,
substituted or unsubstituted, purine or pyrimidine base. Such
purine and pyrimidine bases include, but are not limited to,
natural purines and pyrimidines such as adenine, cytosine, thymine,
guanine, uracil, or other purines and pyrimidines, such as
isocytosine, 6-methyluracil, 4,6-di-hydroxyprimidine, hypoxanthine,
xanthine, 2,6-diaminopurine, 5-azactosine, 5-methyl cystosine, and
the like.
In general, the oligonucleotide includes at least two, preferably
at least 5, and most preferably from 5to 30 nucleotide units.
In one embodiment, the at least one nucleotide unit which includes
the conjugate moiety is the 3' terminal nucleotide unit. In another
embodiment, the at lest one nucleotide unit is the 5' terminal
nucleotide unit.
Alternatively, the at least one nucleotide unit which includes a
conjugate moiety as hereinabove described is one or more nucleotide
units at the 3' end and/or the 5' end of the oligonucleotide. In
yet another embodiment, the at least one nucleotide unit may
alternate with nucleotide units which are unsubstituted (i.e.,
which do not include a conjugate moiety). In another embodiment,
all of the nucleotide units include a conjugate moiety.
The conjugate moiety may be attached to the oligonucleotide at the
purine or pyrimidine base, at the phosphate group, or to the
sugar.
When the conjugate moiety is attached to the base, it is preferably
attached at certain positions of the base, depending upon the base
to which the moiety is attached. When the moiety is attached to
adenine, it may be attached at the C2, N6, or C8 positions. When
the moiety is attached to guanine, it may be attached at the N2 or
C8 positions. When the moiety is attached to cytosine, it may be
attached at the C5 or N4 positions. When the moiety is attached to
thymine or uracil, it may be attached at the C5 position.
The moiety may be attached to a phosphate group at the 5' end, at
an internal position, or at the 3' end of the oligonucleotide. The
moiety may be attached to the 5' end of the oligonucleotide via an
--NH.sub.2 --(CH.sub.2).sub.6 -- linker (such as Aminolink II, for
example), via a phosphodiester linkage, or via other linkers. A
wide variety of linker groups may be employed, depending upon the
nature of the nucleotide unit, the moiety, and whether the linker
group is present during the synthesis of the oligonucleotide. The
linker group may be a single atom, or a functional group. Examples
of linkers include, but are not limited to --NH--, or amino groups,
sulfur atoms, and polyvalent functional groups.
In another embodiment, the linking group is derived from a
polyvalent functional group having at least one atom, and not more
than about 60 atoms other than hydrogen, preferably not more than
about 30 atoms other than hydrogen. The linker group in general has
up to about 30 carbon atoms, preferably not more than about 20
carbon atoms, and up to about 10 heteroatoms, preferably up to
about 6 heteroatoms, and in particular such heteroatoms may be
oxygen, sulfur, nitrogen, or phosphorus. Representative examples of
linker groups include, but are not limited to
--CO--(CH.sub.2).sub.n --NH--;
--CO--(CH.sub.2).sub.n --CO.sub.13 ; ##STR1## ##STR2##
--(CH.sub.2).sub.n --NH--; --CO--; --CO--CH.sub.2 --CH.sub.2
--S--S--; --CH.sub.2 --CH.sub.2 NHCO(CH.sub.2).sub.n CONH--;
--CH.sub.2 CH.sub.2 --NH--Q--(CH.sub.2).sub.n NH--, wherein Q is
2,5-quinondiyl; ##STR3##
In the above structures, n is from 1 to 20, preferably from 2 to
12, and more preferably 6.
The moiety may be attached at internal positions to a phosphate
group via a P--N linker, a P--S linker, or a P--O linker. The
moiety also may be attached at the 3' end to a phosphate group via
a phosphodiester linkage.
When the moiety is attached to a sugar of an oligonucleotide, the
moiety may be attached at the 2'- position; at the hydroxy group of
the 3' -end or the 5' end; via a 3'-terminal ribose dialdehyde; at
the 1'-position; or by using a 3'-terminal amino linker, such as,
for example, a 3'-amino modified controlled pore glass (C3 CPG)
which is commercially available from Glen Research, Serling,
Va.
Although the moieties may be attached to the oligonucleotides at
the various positions and by the various means hereinabove
described, it is to be understood that the scope of the present
invention is not to be limited to such means of attachment.
Amino acids which may be conjugated to the at least one nucleotide
unit include, but are not limited to, alanine, methionine, leucine,
isoleucine, and lysine. The amino acid may be attached to the at
least one nucleotide unit by the acid functionality, the amino
group, or by the side chain.
In one embodiment, an active ester derivative of
fluorenyl-methoxycarbonyl (F-moc)-alanine is reacted with an
oligonucelotide having a 5'-amino group to provide an intermediate
which, after deprotection by treatment with base, provides a
conjugate with the amino acid attached to the oligonucleotide via
its carboxyl group.
In another embodiment, reaction of an oligonucleotide possessing a
5'-amino group with a bifunctional linker arm reagent such as
disuccinimidyl suberate (DSS) provides an activated intermediate
which may react with the amino groups of an amino acid to provide
the desired conjugate.
Other crosslinking agents with shorter or longer linker arms may be
used in place of discuccinimidyl suberate. Alternatively, active
esters can be reacted with the oligonucleotide 2'-hydroxyl group or
a 2'-thiol group to give conjugates linked via ester or thioester
linkages, respectivley. Attachment to the side chain of an amino
acid depends upon the nature of the amino acid because the side
chains differ widely. Attachment to acidic or basic side chains can
be carried out by methods similar to those described for the
carboxyl and amino groups, whereas attachment to hydrocarbon side
chains necessitates introduction of new attachment sites. Both D-
and L-amino acids can be attached by these methods.
Dipeptide mimics which may be conjugated to the at least one
nucleotide unit include, but are not limited to, amino ethyl
glycine, and cephalosporins. Cephalosporins which may be employed
include, but are not limited to, cephalexin, cephradine, cefaclor,
cefadroxil, cefazolin, and cefotiam.
The dipeptide mimic amino ethyl glycine may be reacted with
trifluoroacetic anydride, or (CF.sub.3 CO).sub.2 O, and the
reaction product is then reacted with N-hydroxysuccinimide (NHS)
and dicyclohexyl carbodiimide (CDD). This product is then reacted
with an oligonucleotide having an aminoalkyl linker such as an
NH.sub.2 --(CH.sub.2).sub.6 -- linker attached to the 5'-phosphate
moiety. The amino ethyl glycine attaches to the --NH.sub.2 -- group
of the linker via the carboxyl group. After conjugation, the
trifluoroacetyl protecting groups are removed by treatment with
dilute ammonium hydroxide.
Aminocephalosporins have been shown to be transported into cells
via the dipeptide transport system of intestinal brush border
membranes as was describe din the Journal of Biological Chemistry,
Volume 261, pgs. 14130-14134 (1986), and thus can be considered as
dipeptide mimics. These can be attached to oligonucleotides to
produce conjugates which can be taken up by dipeptide transport
systems and thus be internalized more efficiently. These molecules
possess amino and carboxyl groups, both of which can be used as
attachment sites for conjugation using methods outlined above.
Attachment to the amino group of the cephalosporin can be
accomplished by using a crosslinking agent which forms amide bonds
with both the cephalosporin and with an oligonucleotide amino
group. Attachment via the carboxyl group can be accomplished by
activation to give an active ester which can be reacted directly
with an oligonucleotide amino group, or reacted with an amino group
attached to the 5' or 3'-position via a linker arm. Alternatively,
such active esters can be reacted with the oligonucleotide
2'-hydroxyl group or the 2'-thiol group to give conjugates linked
via ester or thioester linkages, respectivley. In addition, thee
compounds have other ring substituent groups which can be used as
linkage sites without interfering with the amino and carboxyl
functionalities.
Dipeptide mimics such as amino ethyl glycine also possess amino and
carboxyl groups, both of which can be used for attachment.
Attachment via the amino group can be accomplished by using a
crosslinking agent such as disuccinimidyl suberate, which forms
amide bonds with both the dipeptide mimic and with a
2'-oligonucleotide amino group. Other crosslinking agents can also
be employed in place of disuccinimidyl suberate. Attachment via the
carboxyl group can be accomplished by activation using an active
ester as is hereinabove described with respect to amino acids. Such
esters could be reacted directly with a 5'-amino group on the
oligonucleotide, or reacted with an amino group attached to the
5'-position via a linker arm. Alternatively, such active esters can
be reacted with the 2'-hydroxyl group or a 2'-thiol group to give
conjugates linked via ester or thioester linkages,
respectivley.
Sugars which may be conjugated to the at least one nucleotide unit
include, but are not limited to, 5-carbon sugars and 6-carbon
sugars. 5-carbon sugars which may be employed include, but are not
limited to, ribose, arabionse, xylose, and lyxose. 6-carbon sugars
include, but are not limited to, glucose, galactose, mannose,
allose, glucose, idose, talose, and altrose. Preferred sugars are
glucose, galactose, and mannose.
Sugars have several hydroxyl groups which can be used for
attachment to oligonucleotides. In one embodiment, one may react a
partially protected sugar derivative such as
1,2,34-tetraacetyl-D-glucopyranose with a 5'-phosphorylated
oligonucleotide using a condensing agent such as
dicyclohexylcarbodiimide or tri-isopropylbenzenesulfonyl chloride.
Another approach is to use a crosslinking agent to attach a linker
arm bearing an active ester group to a 5'-oligonucleotide amino
group. This active ester would be capable of reacting with a
hydroxyl group of the sugar to give an ester linkage. Yet another
approach is to couple an active ester of an acid derivative of a
sugar to an oligonucleotide amino group to produce an amide
linkage. Still another approach is to react a sugar isothiocyanate
such as 2,3,4,6-tetraacetyl-D-glucopyranose-1-isothiocyanate with a
5' or 3'-oligonucleotide amino group to give a thiourea
linkage.
In another embodiment, the sugar may be protected with one or more
acetyl groups and a phosphate group. The protected sugar is then
reacted with a partially protected oligonucleotide attached to
controlled pore glass as synthesized by using a DNA synthesizer
machine. The protected sugar and the protected oligonucleotide are
reacted in the presence of a coupling agent, which may be
dicyclohexylcarbodiimide or mesitylenesulfonyl chloride to form an
oligonucleotide to which is attached a sugar through attachment of
the sugar to the phosphate group. The protecting groups are
subsequently removed using ammonium hydroxide.
Sugar phosphates can be used as conjugate groups to enhance the
delivery of oligonucleotides into cells. Distler et al., in the
Journal of Biological Chemistry, Vol. 266, pages 21687-92 (1991)
have shown that oligosaccharides containing a terminal
mannose-6-phosphate residue inhibited the binding of
beta-galactosidase to mannose-6-phosphate cell surface receptors
from bovine testis, with the di- and trisaccharides being more
effective inhibitors than the monosaccharide mannose-6-phosphate.
The nature of the penultimate glycosidic linkages of the
oligosaccharides played little or no role in the inhibition of
binding. Tomoda et al., in Carbonhydrate Research, Vol. 213, pages
37-46 (1991) prepared conjugates of mannose-6-phoshpate and
oligosaccharides thereof with bovine serum albumin, and showed that
these conjugates bound to the mannose-6-phosphate receptor from
rabbit alveolar macrophages.
Sugar phosphates which may be attached to the at least one
nucleotide unit include, but are not limited to, manosaccharides
such as mannose-6-phosphate, glucose-6-phosphate,
galactose-6-phosphate, mannose-1-phosphate, glucose-1-phosphate and
galactose-1-phosphate, disaccharides such as
6-O-phosphoryl-.alpha.-D-mannopyranosyl-(1-2)-D-mannopyranose,
6-O-phosphoryl-.alpha.-D-mannopyranosyl-(1-3)-mannopyranose,
6-O-phosphoryl-.alpha.-D-mannopyranosyl-(1-6)-D-mannopyranose,
trisaccharides such as
6-O-phosphoryl-.alpha.-D-mannopyranosyl-(1-2)-D-mannopyranose-(1-2)-D-mann
opyranose, and higher linear or branched oligosaccharides such as
pentamannose-6-phosphate.
Sugar phosphates may be attached to oligonucleotides by a reductive
amination procedure similar to the method used by Baba, et al., in
Carbonhydrate Research, Vol. 177, pages 163-172 (1988) for the
conjugation of pentamannosyl-6-phosphate to bovine serum albumin,
or by formation of a glycoside possessing a linker arm which can be
attached to an oligonucleotide.
Sugar phosphates may be attached to oligonucleotides by
condensation reactions via the phosphate group. For example,
reaction of 1,2,3,4-tetraacetyl-glucose-6-phosphate with the
5'-terminal amino group of an oligonucleotide using a condensing
agent such as tri-isopropylbenzenesulfonyl tetrazolide will produce
a conjugate in which the sugar phosphate is linked to the
oligonucleotide via a phosphoramidate linkage. Alternatively,
1,2,3,4-tetraacetyl=glucose-6-phosphate can be coupled to the
2'-hydroxyl group of a ribonucleotide using the same condensing
agent to produce a conjugate linked via a phosphodiester.
Neurotransmitters which may be conjugated to the at least one
nucleotide unit of the oligonucleotide include, but are not limited
to, dopamine, acetylcholine, epinephrine, norepinephrine, and
serotonin.
Acetylcholine may be attached to the oligonucleotide by reacting
choline, which has the following structural formula: ##STR4##
with (BrCH.sub.2 --CO).sub.2 O to form the following protected
compound: ##STR5##
This compound is reacted with an oligonucleotide having a thiol
group at the 5' end of the oligonucleotide to form an
oligonucleotide to which acetylcholine is conjugated through a
--CO--CH.sub.2 --S-group.
Norepinephrine, dopamine, and serotonin, each of which have an
amino group, may be conjugated to an oligonucleotide by reacting
the amino group with an activated oligonucleotide. Such reaction of
the amino group with the activated oligonucleotide results in
conjugation of the neurotransmitter with the oligonucleotide.
Polymers which may be conjugated with the oligonucleotides in
accordance with the present invention include polyamines,
cyclodextrins, dextrans, polyethyleneimine, polymaleic anhydride,
poly-hydroxypropylmethacrylamide (HPMA), and starches.
Polyamines which may be conjugated to the at least one nucleotide
unit include the naturally occurring cationic compounds sperimine
and spermidine. It is to be understood, however, that the scope of
the present invention is not to be limited to these specific
polyamines. Polyamines can be attached to the at least one
nucleotide unit by methods which include, but are not restricted
to, the following:
1. Reaction of the polyamine with an oligonucleotide having a 5'-
or 3'-phosphomonoester group in the presence of DCC to produce a
conjugate linked by a phosphoramidate linkage.
2. Reaction of the polyamine with an oligonucleotide H-phosphonate
to produce a conjugate in which the polyamine is attached to the
pohosphate backbone.
3. Treatment of an oligonucleotide containing a 3'-terminal
ribonucleotide with periodate to give a dialdehyde which is then
reacted with the polyamine to form a conjugate attached via a
morpholine ring.
In one embodiment, dextran is conjugated with the oligonucelotide
by reacting an activated dextran with a protected oligonucleotide.
Dextran may be activated by reacting dextran with
Br--(CH.sub.2).sub.5 --COOH in the presence of sodium hydroxide to
form dextran-O--(CH.sub.2).sub.5 --COOH, which is then reacted in
the presence of dimethylformamide, hexamethylphosphoramide (HMPA),
pyridine, and DCC to form the active ester derivative: ##STR6##
The preparation of the derivative is described in Pietta, et al.,
Preparative Biochemistry, Vol. 14, pgs. 313-329 (1984). Other
methods for the derivatization of dextran are described in Schact,
Industrial Polysaccharides: Genetic Engineering, Structure/Property
Relations and Applications, Yalpani, ed., pags. 389-400 (1987), and
in Yalpani, et al., Journal of Polymer Science: Polymer Chemistry
Edition, Vol. 23, pgs. 1395-1405 (1985).
This active ester derivative of dextran is reacted with an
oligonucleotide containing an amino group in the presence of sodium
phosphate buffer (pH 8.25) to form an oligonucelotide conjugated
with dextran.
Poly HPMA may be conjugated with an oligonucelotide by reacting a
copolymer of HPMA and the 4-nitrophenyl ester of
N-methacryloylaminocaproic acid with an oligonucleotide protected
with an amino group.
The 4-nitrophenyl ester of N-methacryloylaminocaproic acid is
prepared by reacting methacryloyl chloride with 6-aminohexanoic
acid in the presence of sodium hydroxide, water, and hdyrochloric
acid to form: ##STR7##
This is reacted with 4-nitrophenol in the presence of DCC and
dimethylformamide to form the 4-nitrophenyl ester of
N-methacryloylaminoacaproic acid, which has the following
structure: ##STR8##
HPMA monomer is reacted with the 4-nitrophenyl ester of
N-methacryloylamiocaproic acid in the presence of
2,2'-azobisisobutyronitrile (AIBN) and aceotne at 50.degree. C. to
form a copolymer having the following structure: ##STR9##
Y is from about 10 to about 50, Z is at least 1 (preferably 1), and
n is from 1 to 10.
The details of the formation of this copolymer are described by
Kopecek, et al., in the Journal of Polymer Science, Polymer
Symposium 66, pgs. 15-32 (1979).
This copolymer is than reacted with an oligonucleotide protected
with an amino group to form an oligonucleotide conjugate having the
following structure: ##STR10##
Y, Z and n are as hereinabove described.
Because cyclodextrins and starches possess similar or identical
carboxyhdrate monomer units to those contained in dextran, the same
methods for the conjugation to oligonucleotides can be employed. In
addition, Tabushi, et al., in the Journal of the American Chemical
Society, Vol. 98, pg. 7855 (1976) have described a disulfonate
derivative of .beta.- cyclodextrin which can be reacted with a
diamine to give a .beta.-cyclodextrin-diamine adduct. This latter
derivative can be coupled to an oligonucleotide in a variety of
ways to give a .beta.-cyclodextrin-oligonucleotide conjugate. For
example, reaction of the .beta.- cyclodextrin-diamine adduct with
an oligonucleotide containing a 5'- phosphate group in the presence
of a carbodiimide coupling agent would produce a 5'- linked
conjugate.
Polyethyleneimine (PEI) can be conjugated to oligonucleotides in a
variety of ways. For examples, reaction of PEI with an
oligonucleotide containing a 5'-phosphate group in the presence of
a carbodiimide coupling agent would produce a PEI-oligonucleotide
conjugated at the 5'-position.
The oligonucleotides of the present invention may be employed to
bind to RNA sequences by Watson-Crick hybridization, and thereby
block RNA processing or translation. For example, the
oligonucleotides of the present invention may be employed as
"antisense" complements to target sequences of mRNA in order to
effect translation arrest and selectively regulate protein
production.
The oligonucleotides of the present invention may be employed to
bind double-standard DNA to form triplexes, or triple helices. Such
triplexes inhibit the replication or transcription of DNA, thereby
disrupting gene replication or transcription. Such triplexes may
also protect DNA binding sites from the action of enzymes such as
DNA methylases.
The oligonucleotides of the present invention may be employed to
bind specifically to target proteins, or to selected regions of
target proteins so as to block function or to restore functions
that had been lost by a protein as a result of mutation. For
example, the oligonucleotides of the present invention may be used
to block the interaction between a receptor and its ligand(s) or to
interfere with the binding of an enzyme or its substrate or
cofactor or to interfere otherwise with the catalytic action of an
enzyme. Conversely, the oligonucleotides of the present 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.
The RNA, DNA, or protein target of interest, to which the
oligonucleotide binds, may be present in or on a porkaryotic or
eukaryotic cell, a virus, a normal cell, or a neoplastic cell, in a
bodily fluid or in stool. The target nucleic acids or proteins may
be of plasmid, viral, chromosomal, mitochondrial or plastid origin.
The target sequences may include DNA or RNA open reading frames
encoding proteins, mRNA, ribosomal RNA, snRNA, hnRNA, introns, or
untranslated 5'- and 3'-sequences flanking DNA or RNA open reading
frames. The modified oligonucleotide may therefore be involved in
inhibiting production or function of a particular gene by
inhibiting the expression 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 be used in vitro or in vivo for modifying
the phenotype of cells, or for limiting the proliferation of
pathogens such as viruses, bacteria, portists, Mycoplasma spcies,
Chlamydia or the like, or for killing or interfering with the
growth of neoplastic cells or specific classes of normal cells.
Thus, the oligonucleotides may be administered to a host subject in
a diseased state to inhibit the transcription and/or expression 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 organisms, Giardia organisms, Entamoebas, neoplastic
cells, such as carcinoma cells, sarcoma cells, and lymphoma cells;
specific B-cells; specific T-cells, such as helper cells,
suppressor cells, cytotoxic T-lymphocytes (CTL), natural killer
(NK) cells, etc.
The oligonucleotides may be selected so as to be capable of
interfering with transcription product maturation or production of
proteins by any of the mechanisms involved with the binding of the
subject composition to its target sequence. These mechanism may
include interference with processing, inhibition of transport
across the nuclear membrane, cleavage by endonucleases, or the
like.
The oligonucleotides may be complementary to such sequences as
sequences expressing growth factors, lymphokines, immunoglobulins,
T-cell receptor sites, MHC antigens, DNA or RNA polymerases,
antibiotic resistance, multiple drug resistance (mdr), genes
involved with metabolic processes, in the formation of amino acids,
nucleic acids, or the like, DHFR, etc. as well as introns or
flanking sequences associated 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
Diseases: Antivirals, Human HIV, HSV, CMV, HPV, VZV infections
Antivirals, Animal Chicken Infectious Bronchitis Pig Transmissible
Gastroenteritis Virus infections Antibacterial, Human Drug
Resistance Plasmids Antiparasitic Agents Malaria Sleeping Sickness
(Trypanosomes) Cancer Direct Anti-Tumor Oncogenes and their
products Agents Adjunctive Therapy Drug Resistance genes and their
products Auto Immune Diseases T-cell receptors Rheumatoid Arthritis
Type I Diabetes Systemic Lupus Multiple sclerosis Organ Transplants
OKT3 cells causing GVHD
The oligonucleotides of the present invention may be employed for
binding to target molecules, such as, for example, proteins
including, but not limited to, ligands, receptors, and or enzymes,
whereby such oligonucleotides inhibit the activity of the target
molecules, or restore activity lost through mutation or
modification of the target molecules.
The oligonucleotides of the present invention are administered in
an effective binding amount to an RNA, a DNA, a protein, road
peptide. Preferably, the oligonucleotides are administered to a
host, such as a human or non-human animal host, so as to obtain a
concentration of oligonucelotide in the blood of from about 0.1 to
about 100 .mu.mole/l. It is also contemplated that the
oligonucleotides may be administered in vitro or ex vivo as well as
in vivo.
The oligonucleotides may be administered in conjunction with an
acceptable pharmaceutical carrier as a pharmaceutical composition.
Such pharmaceutical compositions may contain suitable excipients
and auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. Such
oligonucleotides may be administered by intramuscular,
intraperitoneal, intraveneous, or subdermal injection in a suitable
solution. Preferably, the preparations particularly those which can
be administered orally and which can be used for the preferred type
of administration, such as tablets, dragees and capsules, and
preparations which can be administered rectally, such as
suppositories, as well as suitable solutions for administration
parentally or orally, and compositions which can be administered
buccally or sublingually, including inclusion compounds, contain
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.
The pharmaceutical preparations of the present invention are
manufactured in a manner which is itself well known in the art. For
example, the pharmaceutical preparations may be made by means of
conventional mixing, granulating, degree-making, dissolving or
lyophilizing processes. The process to be used will depend
ultimately on the physical properties of the active ingredient
used.
Suitable excipients are, in particular, fillers such as sugar, for
example, lactose or sucrose, mannitol or sorbiotl, cellulose
preparations and/or calcium phosphates, for example, tricalcium
phosphate or calcium hydrogen phosphate, as well as binders such as
starch or paste, using, for example, maize starch, wheat starch,
rice starch, potato starch, gelatin, gum tragacanth, methylc
ellulose, hydroxypropylmethylcellulose, sodium
carboxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinyl pyrrolidone. If desired, disintegrating agents may
be added, such as the above-mentioned starches as well as
carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or
aliginic acid or a salt thereof, such as sodium aliginate.
Auxiliaries are flow-regulating agents and lubricants, such as, for
example, silica, talc, stearic acid or salts thereof, such as
magnesium stearate or calcium stearate, and/or polyethylene glycol.
Dragee cores may be provided with suitable coatings which, if
desired, may be resistant 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, solutions of suitable cellulose
preparations such a acetylcellulose phthalate or
hydroxypropylmethylcellulose phthalate, are used. Dyestuffs and
pigments may be added to the tablets of dragee coatins, for
example, for identification or in order to characterize different
combinations of active compound loses.
Other pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer such as glycerol or sorbitol. The
push-fit capsules can contain the oligonucleotide in the form of
granules which may be mixed with fillers such as lactose, binders
such as starches, and/or lubricants such as talc or magnesium
stearate and, optionally, stablizers. In soft capsules, the active
compounds are preferably dissolved or suspended in suitable
liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycols. In addition, stabilizers may be added.
Possible pharamecutical preparations which can be used rectally
include, for example, suppositories, which consist of a combination
of the active compounds with a suppository base. Suitable
suppository bases are, for example, natural or synthetic
triglycerides, paraffin hydrocarbons, polyethylene glycols, or
higher alkanols. In addition, it is also possible to use gelatin
rectal capsules which consist of a combination of the active
compounds with a base. Possible base materials include, for
example, liquid triglycerides, polyethylene glycols, or paraffin
hydrocarbons.
Suitable formulations for parenteral administration include aqueous
solutions of the active compounds in water-soluble or
water-dispersible form. In addition, suspensions of the active
compounds as appropriate oil injection suspensions may be
administered. Suitable lipophilic solvents or vehicles include
fatty oils, for example, sesame oil, or synthetic fatty acid
esters, for example, ethyl oleate or triglycerides. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension including, for example, sodium
carboxymethyl cellulose, sorbitor and/or dextran. Optionally, the
suspension may also contain stabilizers.
Additionally, the compounds of the present invention may also be
administered encapsulated in liposomes, wherein the active
ingredient is contained either dispersed or variously present in
corpuscles consisting of aqueious concentric layers adherent to
lipidic layers. The active ingredient, depending upon its
solubility, may be present both in the aquious layer, in the
lipidic layer, or in what is generally termed a liposomic
suspension. The hydrophobic layer, generally but not exclusively,
comprises phospholipids such as lecithin and sphingomycelin,
steroids such as cholesterol, surfactants such as dicetylphosphate,
stearylamine, or phsophatidic acid, and/or other materials of a
hydrophobic nature. The diameters of the liposomes generally range
from about 15 nm to about 5 microns.
The oligonucleotide conjugates of the present invention may also be
employed as diagnostic probes. In this approach the conjugate group
serves as a radioactive or nonradioactive reporter group for the
detection of nucleic acid sequences of interest. In one embodiment,
the DNA-containing sample to be analyzed is immobilized on an inert
solid support such as a nitrocellulose membrane and then annealed
with the oligonucleotide conjugate. This annealing procedure allows
the conjugated oligonucleotide to bind to the DNA provided that the
base sequences are complementary to each other. After a series of
washing steps the duplex of the oligonucleotide conjugate with the
DNA is exposed to a complex of an antibody to the conjugate group
attached to an enzyme such as alkaline phosphatase. After a further
series of washing steps, the DNA-antibody-enzyme complex is
detected by exposure to a chromogenic substrate which generates a
purple-blue color in the area of the bound complex.
In another embodiment, the conjugate group on the oligonucleotide
may be a carbonhydrate and the modified oligonucleotide bound to
the complementary DAN can be detected by complex formation with a
lectin specific for the carbohydrate conjugate group, such lectin
subsequently being complexed with an antibody attached to an enzyme
such as alkaline phosphatase.
Several other conjugate groups have ben used in this manner. For
example, oligonucleotide diagnostic probes have been prepared by
attachment of digoxigenin to the 5-position of pyrimidine bases are
reported by Muhlegger et al. in Nucleosides and Nucleotides, Vol.
8, pages 1161-1163 (1989), and conjugates of the 2,4-dinitrophenyl
group have been reported by Vincent et al. in Nucleic Acids
Research, Volume 10, pages 6787-6796 (1982).
The oligonucleotide conjugates can also be used as diagnostic
probes to interact with RNA's in a sample provide that the target
RNA has a sequence complementary to the sequence of the conjugated
oligonucleotide. If both DNA and RNA are present in the sample and
it is desired to measure only DNA, the sample can be treated with
RNase prior to addition of the oligonucleotide conjugate. If it is
desired to measure only RNA, the sample can be treated with DNase
prior to addition of the oligonucleotide conjugate. The
oligonucleoitde conjugates can also be used as diagnostic probes to
interact with proteins in a sample provided that the target protein
binds tightly or specifically to the conjugated oligonucleotide
because of the sequence of the conjugated oligonucleotide. For
example, the glucocorticoid receptor protein has been demonstrated
to bind with high affinity to the sequence GGTACAN.sub.3 TGTTCT,
(SEQ ID NO: 1) wherein N is any purine or pyrimidine base. (R. M.
Evans, Science, Vol. 240, pgs. 889 (1988)). A double-stranded
oligonucleotide in which one strand is an oligonucleotide conjugate
of the present invention could be used as a diagnostic probe to
measure glucocorticoid receptor protein in a sample. Other
DNA-binding proteins can be similarly measured. Bock et al.
(Nature, Vol. 355, pages 564-566, (1992) have demonstrated that the
protein thrombin binds tightly to DNA oligonucleotides containing
the consensus sequence GGTTGG(N.sub.3)GGTTGG. (SEQ ID NO: 2) An
oligonucleotide of the present invention containing ribonucleotides
or deoxyribonucleotides of a given sequence might thus be used to
detect a protein which bind that sequence.
The invention will now be described with respect to the following
examples; however, the scope of the present invention is not
intended to be limited thereby.
EXAMPLE 1
Attachment of Methionine to an Oligonucleotide
A 15-base oligonucleotide was prepared on a DNA synthesizer using
standard reagents as supplied by the manufacturer.
Cyanoethyoxy-di-isopropylamin-trifluoroacetylaminohexyloxy-phosphine
(Aminolink II) was used for the final coupling step to introduce a
linker arm onto the 5'-terminus of the oligonucleotide. The crude
oligonucleotide was treated with concentrated ammonium hydroxide
for 12 hours at 55.degree. C. to remove the base protecting groups,
and the solution was evaporated to dryness. A solution of the
5'-amino oligonucleotide (190 OD.sub.260 units) in 0.1 M sodium
bicarbonate (pH 8.2; 150 .mu.l) was added to an Eppendorf tube
containing a solution of the N-hydroxysuccinimide ester of
N-fluoroenylmethoxycarbonyl-L-methionine (6.2 mg) in
dimethylsulfoxide (300 .mu.l), and the resulting mixture was
vortexed for 15 sec. and then incubated at ambient temperature for
7 hours. The product was neutralized by addition of acetic acid to
pH 7, stored overnight, and evaporated to dryness. The residue was
coevaporated with water (3.times.25 ml), dissolved in water (6 ml)
and lyophilized overnight. The residue was then purified by
preparative reversed phase C.sub.18 HPLC (9.times.25 cm) using a
gradient of 0.1 M triethylammonium acetate (pH 7.1)/acetonitrile as
eluent. The fraction containing the desired product was collected,
evaporated to dryness and coevaporated with water (3.times.25 ml).
The residue was treated with a solution of morpoholine in water
(1:1, 2 ml) for 45 min. at room temp to remove the
fluorenylmethoxycarbonyl group, evaporated to dryness and
coevaporated with water (3.times.1 ml) to remove traces of
morpholine. The residue was partitioned between water and ethyl
acetate (2 ml each) and the aqueous layer was extracted with ethyl
acetate (2.times.2 ml) and lyophilized to dryness. The residue was
dissolved in water (1 ml) and converted into the sodium salt by
passage through a column of ion exchange resin (Dowex AG 50W-X8,
sodium form, 0.7.times.6 cm). The eluent was collected and
evaporated to dryness to give the oligonucleotide-methionine
conjugate.
EXAMPLE 2
Attachment of Glucose to a 5'-Amino-oligonucleotide
A solution of the 5'-amino oligonucleotide (100 OD.sub.260 units)
in 0.2 M sodium phosphate (pH 7.0; 70 .mu.l) is added to an
Eppendorf tube containing a solution of
2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyransoyl-isothiocyanate (1
mg) in dimethylformamide (30 .mu.l) and the resulting mixture is
stirred for 15 seconds and then incubated at ambient temperature
for 1 day. The mixture is deprotected with aqueious ammonium
hydroxide at room temperature and evaporated to dryness. The
residue is dissolved in water, filtered, and purified by reversed
phase C.sub.18 HPLC to give the glucose-oligonucleotide
conjugate.
EXAMPLE 3
Synthesis of an Oligonucleotide-Aminoethyl Glycine Conjugate
A solution of aminoethyl glycine in pyridine is treated with
trifluoroacetic anhydride at 0.degree. C. For 18 hours. Water is
added to the chilled solution, and after 1 hour the mixture is
evaporated to dryness and pumped in vacuo overnight to give the N,
N-trifluoroacetyl derivative of aminoethyl glycine. This material
is dissolved in dimethylformamide and treated with a solution of
5'-amino oligonucleotide in acetate buffer, pH 5 followed by
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC). After
incubation for 18 hours at room temperature, the solution is
evaporated to dryness, dissolved in N-ammonium hydroxide, and
stored at room temperature overnight. The residue is dissolved in
0.02 M triethylammonium bicarbonate and purified by preparative
reversed phase C.sub.18 HPLC to give the aminoethyl
glycine-oligonucleotide conjugate.
EXAMPLE 4
Synthesis of an Oligonucleotide-Cephalosporin Conjugate
The aminocephalosporin Cephalexin (Shionogi and CO.) is dissolved
in pyridine and treated with trifluoroacetic anhydride at 0.degree.
C. for 18 hours. Water is added to the chilled solution, and after
1 hour the mixture is evaporated to dryness and pumped in vacuo
overnight, dissolved in dimethylformamide and treated with a
solution of 5'-amino oligonucleotide in acetate buffer, pH 5,
followed by addition of EDC. After storage for 18 hours at room
temperature, the solution is evaporated to dryness, dissolved in
N-ammonium hydroxide, and incubated at room temperature overnight.
The residue is filtered, dissolved in 0.02 M triethylammonium
bicarbonate and purified by preparative reversed phase C.sub.18
HPLC to give the aminocephalosporin-oligonucleotide conjugate.
EXAMPLE 5
Attachment of Glucose-6-Phosphate to an Oligonucleotide
A suspension of glucose-6-phosphate, pyridinium salt, in pyridine
is treated with stirring at 0.degree. C. with acetic anhydride for
18 hours, and then treated with water for 4 hours. The solvents are
removed by evaporation and the residue is dissolved in
dimethylformamide and treated with a solution of 5'-amino
oligonucleotide in acetate buffer, pH 5 followed by EDC. After
incubation for 18 hours at room temperature, the solution is
evaporated to dryness, dissolved in 5 N ammonium hdyroxide, and
stored at room temperature overnight. This solution is evaporated
to dryness, and the residue is dissolved in 0.02 M triethylammonium
bicarbonate, filtered, and the filtrate is purified by preparative
reverse phase C.sub.18 HPLC to give the
glucose-6-phosphate-oligonucleotide conjugate.
EXAMPLE 6
Preparation of a Neurotransmitter-Oligonucleotide Conjugate
An oligonucleotide is prepared on a DNA synthesizer using standard
reagents as supplied by the manufacturer.
Cyanoethoxy-di-isopropylamino-trifluoroacetylaminohexyloxy-phosphine
(Aminolink II) is used for the final coupling step to introduce a
linker arm onto the 5'-terminus of the oligonucleotide. The crude
oligonucleotide is treated with concentrated ammonium hydroxide for
12 hours at 55.degree. C. to remove the protecting groups, and the
solution is evaporated to dryness. A solution of the 5'-amino
oligonucleotide in 0.1 M sodium bicarbonate buffer (pH 8.2) is
treated with a solution of dihydroxysuccinimidyl suberate (DSS) in
dimethylsulfoxide for 15 minutes at room temperature and then
applied to a column of Sephadex G25. The column is eluted with
water, and the eluent is monitored by UV spectroscopy. The
fractions containing the first peak are combined, frozen as quickly
as possible, and lyophilized to dryness. The residue is redissolved
in bicarbonate buffer and immediately treated with a solution of
serotonin in dimethylsulfoxide for 18 hours at room temperature.
The solution is then applied to a Sephadex G25 column which is
eluted with water, and the first UV absorbing peak is collected,
partially evaporated, and purified by reverse phase C18 HPLC. The
later eluting peak is collected and lyophilized to give the
serotonin-oligonucleotide conjugate.
EXAMPLE 7
Synthesis of a Poly(hydroxypropyl-methacrylamide)-Oligonucleotide
Conjugate
A copolymer of N-(2-hydroxypropyl) methacrylamide and
N-methacryloyl-6-aminocaproyl-p-nitrophenyl ester, prepared by the
method of Kopecek and Rejmanova in the Journal of Polymer Science:
Polymer Symposium, Vol. 66, pp. 15-32 (1979) is dissolved in
dimethylsulfoxide and added to a solution of a
5'-amino-oligonucleotide in aqueous bicarbonate buffer. The
solution is incubated at room temperature for 18 hours, evaporated
to remove solvent, and redissolved in water. The solution is
centrifuged to remove solid and the supernatant is purified on a
reverse phase C18 HPLC column. The late eluting peak is evaporated
to dryness to give the poly-HPMA-oligonucleotide conjugate.
EXAMPLE 8
Synthesis of a Dextra-Oligonucleotide Conjugate
A 15-base oligonucleotide was prepared on a DNA synthesizer using
standard reagents as supplied by the manufacturer.
Cyanoethoxy-di-isopropylamino-trifluoroacetylaminohexyloxy-phosphine
(Aminolink II) was used for the final coupling step to introduce a
linker arm onto the 5'-terminus of the oligonucleotide. The crude
oligonucleotide was treated with concentrated ammonium hydroxide
for 12 hours at 55.degree. C. to remove the protecting groups, and
the solution was evaporated to dryness. The residue was dissolved
in water (0.5 ml) and passed through a Sephadex G25 column which
was eluted with water. Fractions were monitored by UV and the first
peak to be eluted was evaporated to dryness and dissolved in 0.2 M
sodium phosphate buffer pH 8.25. The solution was added to a sample
of carboxy-dextran-N-hydroxysuccinimide ester (40 mg) which was
prepared by the method of Pietta et al., Preparative Bochemistry,
Vol. 14, pp. 313-329 (1984). The reactants were stored at room
temperature for two days, diluted to 1 ml with water and dialyzed
against distilled water for 24 hours at 4.degree. C. using a
dialysis membrane with a molecular weight cutoff of 8000. The
product was lyophilized, dissolved in 0.1 M triethylammonium
acetate buffer pH 7.1 (TEAB), and passed through a Sephadex G25
column (1.times.30 cm) using TEAB as the eluent. Fractions of 1 ml
were collected, and fractions 17-24 were combined and evaporated to
dryness. The residue was further purified on a Dionex NucleoPac
anion exchange column (4.times.250 mm) using a gradient of 25 mM
tris chloride pH 8 containing 5% acetonitrile as buffer A, and 25
mM tris chloride, 1 M ammonium chloride, pH 8 containing 5%
acetonitrile as buffer B. Buffer B was increased from 15% at T=0 to
55% at 15 min. The fractions eluting at 7-9.5 min. were combined
and dialyzed against distilled water at 4.degree. C. During this
time the water was changed at regular intervals. The product was
lyophilized to give the dextran-oligonucleotide conjugate.
It is to be understood, however, that the scope of the present
invention is not to be limited to the specific embodiments
described above. The invention may be practiced other than as
particularly described and still be within the scope of the
accompanying claims.
* * * * *