U.S. patent application number 10/601242 was filed with the patent office on 2004-02-26 for gapped 2' modified oligonucleotides.
This patent application is currently assigned to ISIS Pharmaceuticals, Inc.. Invention is credited to Cook, Phillip Dan, Monia, Brett P..
Application Number | 20040038274 10/601242 |
Document ID | / |
Family ID | 25216474 |
Filed Date | 2004-02-26 |
United States Patent
Application |
20040038274 |
Kind Code |
A1 |
Cook, Phillip Dan ; et
al. |
February 26, 2004 |
Gapped 2' modified oligonucleotides
Abstract
Oligonucleotides and other macromolecules are provided that have
increased nuclease resistance, substituent groups for increasing
binding affinity to complementary strand, and sub-sequences of
2'-deoxy-erythro-pentofuran- osyl nucleotides that activate RNase H
enzyme. Such oligonucleotides and macromolecules are useful for
diagnostics and other res arch purposes, for modulating protein in
organisms, and for th diagnosis, detection and treatment of other
conditions susceptible to antisense therapeutics.
Inventors: |
Cook, Phillip Dan; (Vista,
CA) ; Monia, Brett P.; (Carlsbad, CA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE - 46TH FLOOR
PHILADELPHIA
PA
19103
US
|
Assignee: |
ISIS Pharmaceuticals, Inc.
|
Family ID: |
25216474 |
Appl. No.: |
10/601242 |
Filed: |
June 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10601242 |
Jun 20, 2003 |
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09951052 |
Sep 12, 2001 |
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09951052 |
Sep 12, 2001 |
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09453514 |
Dec 1, 1999 |
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6326199 |
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09453514 |
Dec 1, 1999 |
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09144611 |
Aug 31, 1998 |
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6146829 |
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09144611 |
Aug 31, 1998 |
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08861306 |
Apr 21, 1997 |
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5856455 |
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08861306 |
Apr 21, 1997 |
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08244993 |
Jun 21, 1994 |
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5623065 |
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08244993 |
Jun 21, 1994 |
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PCT/US92/11339 |
Dec 23, 1992 |
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08861306 |
Apr 21, 1997 |
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07814961 |
Dec 24, 1991 |
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08861306 |
Apr 21, 1997 |
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08007996 |
Jan 21, 1993 |
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Current U.S.
Class: |
435/6.11 ;
514/44A; 536/23.1 |
Current CPC
Class: |
C07H 21/04 20130101;
C12N 15/1135 20130101; C12N 2310/3521 20130101; A61P 35/00
20180101; C12Q 1/6832 20130101; C12N 2310/3533 20130101; C12N
2310/3527 20130101; C12N 2310/321 20130101; C12Q 1/6816 20130101;
C12N 2310/32 20130101; C12N 2310/3531 20130101; C07K 14/003
20130101; C12Q 1/6813 20130101; C07H 21/00 20130101; C12Q 2525/125
20130101; A61P 31/00 20180101; C12N 2310/322 20130101; A61P 43/00
20180101; C07K 14/82 20130101; C12N 2310/315 20130101; C12Q 1/6813
20130101; C12Q 2525/125 20130101; C12Q 2521/319 20130101; C12Q
1/6832 20130101; C12Q 2525/101 20130101; C12Q 1/6816 20130101; C12Q
2525/125 20130101 |
Class at
Publication: |
435/6 ; 514/44;
536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/02; C07H 021/04; A61K 048/00 |
Claims
What is claimed is:
1. An ligonucl otid comprising a s quence of nucleotide units
capable of specifically hybridizing to a strand of nucleic acid,
wherein: at least one of said nucleotide units is functionalized to
increase nuclease resistance of said oligonucleotide; at least one
of said nucleotide units bears a substituent group that increases
binding affinity of said oligonucleotide to said strand of nucleic
acid; and a plurality of said nucleotide units have
2'-deoxy-erythro-pentofuranosyl sugar moieties, said
2'-deoxy-erythro-pentofuranosyl nucleotide units being
consecutively located in said sequence of nucleotide units.
2. The oligonucleotide of claim 1 wherein said substituent group
for increasing binding affinity comprises a 2'-substituent
group.
3. The oligonucleotide of claim 2 wherein said 2'-substituent group
is fluoro, C1-C9 alkoxy, C1-C9 aminoalkoxy, allyloxy,
imidazolealkoxy and poly(ethylene glycol).
4. The oligonucleotide of claim 1 wherein each of said nucleotide
units is a phosphorothioate r phosphorodithioate nucleotide.
5. The oligonucleotide of claim 1 wherein the 3' terminal
nucleotide unit of said oligonucleotide includes a nuclease
resistance modifying group on at least one of the 2' or the 3'
positions of said nucleotide unit.
6. The oligonucleotide of claim 1 wherein: a plurality of said
nucleotide units bear substituent groups that increases binding
affinity of said oligonucleotid to said strand of nucleic acid,
said substituent-bearing nucleotides being divided into a first
nucleotide unit sub-sequence and a second nucleotide unit
sub-sequence; and said plurality of 2'-deoxy-erythro-pentofuranosyl
nucleotide units is positioned in said sequence of nucleotide units
betw n said first nucl otide unit sub-sequence and said second
nucle tide unit sub-sequence.
7. The oligonucleotide of claim 1 wherein: a plurality f said
nucleotide units bear substituent groups that increase binding
affinity of said ligonucleotide to said complementary strand of
nucleic acid; and at least a portion of said substituent-bearing
nucleotide are consecutively located at one of the 3' terminus or
the 5' terminus of said oligonucleotide.
8. The oligonucleotide of claim 1 wherein at least five of said
nucleotide units have 2'-deoxy-erythro-pentofuranosyl sugar
moieties, said at least five 2'-deoxy-erythro-pentofuranosyl
nucleotide units being consecutively located in said sequence of
nucleotide units.
9. The oligonucleotide of claim 1 wherein from ne to about eight of
said nucleotide units bear a substituent group that increases the
binding affinity of said oligonucleotide to said complementary
strand, said substituent-bearing nucleotide units being
consecutively located in said sequence of nucleotide units.
10. The oligonucleotide of claim 1 wherein: from one to about eight
of said nucleotide units bear a substituent group for increasing
the binding affinity of said oligonucleotide to said complementary
strand, said substituent-bearing nucleotide units being
consecutively located in said sequence of nucleotide units; and at
least five of said nucleotide units have
2'-deoxy-erythro-pentofuranosyl sugar moieties, said at least five
2'-deoxy-erythro-pentofuranosyl nucleotide units being
consecutively located in said sequence of nucleotide units.
11. An oligonucleotide comprising a sequence f phosphorothioate
nucleotides capable of specifically hybridizing to a strand of
nucleic acid, wherein: a plurality of said nucleotides bear a
substituent group that increases binding affinity of said
oligonucleotide to said strand of nucleic acid; and a plurality of
said nucleotides have 2'-deoxy-erythro-pent furanosyl sugar moi
ties.
12. Th oligonucleotide of claim 11 wher in said substituent group
for incr asing binding affinity compris s a 2'-substituent gr
up.
13. The oligonucleotide of claim 12 wherein said 2'-substituent
group is fluoro, C1-C9 alkoxy, C1-C9 aminoalkoxy r allyloxy.
14. The oligonucleotide of claim 12 including: a further plurality
of said nucleotides bearing 2'-substituent groups; said
2'-deoxy-erythro-pentofur- anosyl nucleotides being positioned in
said oligonucleotide between groups of nucleotides having said
2'-substituent group located thereon.
15. The oligonucleotide of claim 11 wherein said
substituent-bearing nucleotides are located at one of the 3'
terminus or the 5' terminus of said oligonucleotide.
16. An oligonucleotide comprising a sequence of phosphorothioate
nucleotides capable of specifically hybridizing to a strand of
nucleic acid, wherein: a first portion of said nucleotides have
2'-deoxy-2'-fluoro, 2'-methoxy, 2'-ethoxy, 2'-propoxy,
2'-aminopropoxy or 2'-allyloxy pentofuranosyl sugar moieties; and a
further portion of said nucleotides have
2'-deoxy-erythro-pentofuranosyl sugar moieties.
17. An oligonucleotide of claim 16 wherein said first portion of
said nucleotides are located at either the 3' terminus or the 5'
terminus of said oligonucleotide.
18. An oligonucleotide of claim 17 including: an additional portion
of said nucleotides having 2'-d oxy-2'-fluoro, 2'-methoxy,
2'-ethoxy, 2'-propoxy, 2'-aminopropoxy or 2'-allyloxy
pentofuranosyl sugar moieties; and said further portion of said
nucleotides positioned in said oligonucleotide between said first
portion of nucleotides and said additional portion of said
nucleotides.
19. A method of treating an organism having a diseas characterized
by the undesired production of a protein comprising contacting the
organism with an oligonucleotide having a s quence of nucleotides
capable of specifically hybridizing to a strand of nucleic acid
coding f r said pr tein at least one of th nucl otides being functi
nalized to increase nuclease resistance of the oligonucleotide, a
plurality of the nucl otides having a substituent group located
thereon to increase binding affinity of the oligonucleotide to the
strand of nucleic acid, and a plurality of the nucleotides having
2'-deoxy-erythro-pentofuranosyl sugar moieties.
20. The method of claim 19 wherein each of said nucleotides is a
phosphorothioate nucleotide.
21. The method of claim 19 wherein said substituent group is a
2'-substituent group.
22. The method of claim 21 wherein said 2'-substituent group is
fluoro, alkoxy, aminoalkoxy or allyloxy.
23. A pharmaceutical composition comprising: an pharmaceutically
effective amount of an oligonucleotide having a sequence of
nucleotides capable of specifically hybridizing to a strand of
nucleic acid, at least one of the nucleotides being functionalized
to increase nuclease resistance of the oligonucleotide, a plurality
of the nucleotides having a substituent group located thereon to
increase binding affinity of the oligonucleotide to a complementary
strand of nucleic acid; a plurality of the nucleotides having
2'-deoxy-erythro-pentofuranosyl sugar moieties; and a
pharmaceutically acceptable diluent or carrier.
24. A method of modifying in vitro a sequence-specific nucleic
acid, comprising contacting a test solution containing RNase H and
said nucleic acid with an oligonucleotide having a sequence of
nucleotides capable of specifically hybridizing to a strand of
nucleic acid where at least one of the nucleotides is
functionalized to increase nuclease resistance of the
oligonucleotide, where a plurality of the nucleotides have a
substituent group located thereon to increase binding affinity of
the oligonucleotide to a complementary strand of nucleic acid, and
where a plurality of the nucleotides have
2'-deoxy-erythro-pentofuranosyl sugar moieti s.
25. A method f c ncurrently enhancing hybridizati n and RNase H
activation in a rganism comprising c ntacting the organism with an
olig nucleotide having a sequence of nucleotides capabl of
specifically hybridizing to a complementary strand of nucleic acid
and where at least one of the nucleotides is functionalized to
increase nuclease resistance of the oligonucleotide, where a
plurality of the nucleotides have a substituent group located
thereon to increase binding affinity of the oligonucleotide to a
complementary strand f nucleic acid, and where a plurality of the
nucleotides have 2'-deoxy-erythro-pentofuranosyl sugar moieties
26. A macromolecule comprising a plurality of nucleosides linked by
covalent linkages in a sequence that is hybridizable to a
complementary nucleic acid, wherein: said nucleosides are selected
from .alpha.-nucleosides, .beta.-nucleosides including
2'-deoxy-erythro-pentof- uranosyl .beta.-nucleosides,
4'-thionucleosides and carbocyclic-nucleosides; said linkages are
selected from charged phosphorous linkages, neutral phosphorous
linkages or non-phosphorous linkages; and said sequence of linked
nucleosides contains at least two nucleoside regions, wherein: a
first of said regions includes nucleosides selected from said
a-nucleosides linked by charged and neutral 3'-5' phosphorous
linkages, said .alpha.-nucleosides linked by charged and neutral
2'-5' phosphorous linkages, said .alpha.-nucleosides linked by
non-phosphorous linkages, said 4'-thionucleosides linked by charged
and neutral 3'-5' phosphorous linkages, said 4'-thionucleosides
linked by charged and neutral 2'-5' phosphorous linkages, said
4'-thionucleosides linked by non-phosphorous linkages, said
carbocyclic-nucleosides linked by charged and neutral 3'-5'
phosphorous linkages, said carbocyclic-nucleosides linked by
charged and neutral 2'-5' phosphorous linkages, said
carbocyclic-nucleosides linked by non-phosphorous linkages, said
.beta.-nucleosides linked by charged and neutral 2'-5' linkages,
and said .beta.-nucleosides linked by n n-phosphor us linkages; and
a second of said regions consists of said
2'-deoxy-erythro-pentofuran- osyl .beta.-nucleosides linked by
charged 3'-5' phosphorous linkages having a negative charge at
physiological pH.
27. A macromolecule of claim 26 wherein said sec nd region includes
at least 3 of said 2'-deoxy-erythro-pentofuranosyl
.beta.-nucleosides.
28. A macromolecule of claim 26 wherein said second nucleoside
region is position between said first nucleoside region and a third
nucleoside region, said third nucleosid region including
nucleosides selected from said .alpha.-nucleosides linked by
charged and neutral 3'-5' phosphorous linkages, said
.alpha.-nucleosides linked by charged and neutral 2'-5' phosphorous
linkages, said .alpha.-nucleosides linked by non-phosphorous
linkages, said 4'-thionucleosides linked by charged and neutral
3'-5' phosphorous linkages, said 4'-thionucleosides linked by
charged and neutral 2'-5' phosphorous linkages, said
4'-thionucleosides linked by non-phosphorous linkages, said
carbocyclic-nucleosides linked by charged and neutral 3'-5'
phosphorous linkages, said carbocyclic-nucleosides linked by
charged and neutral 2'-5' phosphorous linkages, said
carbocyclic-nucleosides linked by non-phosphorous linkages, said
.beta.-nucleosides linked by charged and neutral 2'-5' linkages,
and said .beta.-nucleosides linked by non-phosphorous linkages.
29. A macromolecule of claim 26 wherein said charged phosphorous
linkages include phosphodiester, phosphorothioate,
phosphorodithioate, phosphoroselenate or phosphorodiselenate
linkages.
30. A macromolecule of claim 26 wherein said charged phosphorous
linkages is phosphodiester or phosphorothioat.
31. A macromolecule of claim 26 wherein said neutral phosphorous
linkages include alkyl and aryl phosphonates, alkyl and aryl
phosphoroamidit s, alkyl and aryl phosphotriesters, hydrogen
phosphonate and boranophosphate linkages.
32. A macromolecule of claim 26 wher in said non-ph sphor us linkag
s include peptide linkages, hydrazine linkages, hydr xy-amine
linkages, carbamate linkages, morpholine linkages, carbonate
linkages, amide linkages, oxymethyleneimine linkages, hydrazide
linkages, silyl linkages, sulfide linkages, disulfide linkages,
sulfone linkages, sulfoxide linkages, sulfonate linkages,
sulfonamide linkages, formacetal linkages, thioformacetal linkages,
oxime linkages and ethylene glycol linkages.
33. A macromolecule of claim 26 wherein said first nucleoside
region includes at least two .alpha.-nucleoside linked by a charged
or neutral 3'-5' phosphorous linkages.
34. A macromolecule comprising a plurality of units linked by
covalent linkages in a sequence that is hybridizable to a
complementary nucleic acid, wherein: said units are selected from
nucleosides and nucleobases: said nucleosides are selected from
.alpha.-nucleosides, .beta.-nucleosides including
2'-deoxy-erythro-pentofuranosyl .beta.-nucleosides,
4'-thionucleosides, and carbocyclic-nucleosides; said nucleobases
are selected from purin-9-yl and pyrimidin-1-yl heterocyclic bases;
said linkages are selected from charged 3'-5' phosphorous, neutral
3'-5' phosphorous, charged 2'-5' phosphorous, neutral 2'-5'
phosphorous or non-phosphorous linkages; and said sequence of
linked units is divided into at least two regions, wherein: a first
of said regions includes said nucleobas s linked by non-phosphorous
linkages and nucleobas s that are attached to phosphate linkages
via non-sugar tethering groups, and nucleosides selected from said
.alpha.-nucleosides linked by charged and neutral 3'-5' phosphorous
linkages, said .alpha.-nucleosides linked by charged and neutral
2'-5' phosphorous linkages, said .alpha.-nucleosid s link d by
non-phosphorous linkages, said 4'-thionucleosides linked by charged
and neutral 3'-5' phosphorous linkages, said 4'-thionucleosides
linked by charged and neutral 2'-5' ph sphorous linkages, said
4'-thionucleosides linked by non-phosphorous linkages, said
carbocyclic-nucleosides linked by charged and neutral 3'-5'
phosphorous linkages, said carbocyclic-nucleosides linked by
charged and neutral 2'-5' phosphorous linkages, said
carbocyclic-nucleosides linked by non-phosphorous linkages, said
.beta.-nucleosides linked by charged and neutral 2'-5' linkages,
and said .beta.-nucleosides link d by non-phosphorous linkages; and
a second of said regions includes said
2'-deoxy-erythro-pentofuranosyl .beta.-nucleosides linked by
charged 3'-5' phosphorous linkages having a negative charge at
physiological pH.
35. The macromolecule of claim 34 wherein said first region
includes at least two nucleobases linked by a non-phosphate
linkage.
36. The macromolecule of claim 35 wherein said non-phosphate
linkage is a peptide linkage.
37. The macromolecule of claim 35 wherein said second region is
positioned between said first region and a third region, said third
region including said nucleobases linked by non-phosphorous
linkages and nucleobases that are attached to phosphate linkages
via a non-sugar tethering moiety, and nucleosides selected from
said a-nucleosides linked by charged and neutral 3'-5' phosphorous
linkages, said .alpha.-nucleosid s linked by charged and neutral
2'-5' phosphorous linkages, said .alpha.-nucleosides linked by
non-phosphorous linkages, said 4'-thionucleosides linked by charged
and neutral 3'-5' phosphorous linkages, said 4'-thionucleosides
linked by charged and neutral 2'-5' phosphorous linkages, said
4'-thionucleosides linked by non-phosphorous linkages, said
carbocyclic-nucleosides link d by charged and neutral 3'-5'
phosphorous linkages, said carbocyclic-nucleosides linked by
charged and neutral 2'-5' phosphorous linkages, said
carbocyclic-nucleosides linked by non-phosph rous linkages, said
.beta.-nucleosides linked by charged and neutral 2'-5' linkages,
and said .beta.-nucleosides linked by non-phosphorous linkages.
38. A macromolecule of claim 35 wherein said nucleobases are
selected from adenine, guanine, cytosin , uracil, thymine,
xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl
adenine, 2-propyl and other alkyl adenine, 5-halo uracil and
cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo
uracil), 4-thiouracil, 8-halo, amino, thiol, thiolalkyl, hydroxyl
and other 8 substituted adenine and guanine, or 5-trifluoromethyl
uracil and cytosine.
39. A macromolecule comprising a plurality of units linked by
covalent linkages in a sequence that is hybridizable to a
complementary nucleic acid, wherein: said units are selected from
nucleosides and nucleobases; said nucleosides are selected from
.alpha.-nucleosides, .beta.-nucleosides, 4'-thionucleosides and
carbocyclic-nucleosides; said nucleobases are selected from
purin-9-yl and pyrimidin-1-yl heterocyclic bases; said linkages are
selected from charged phosphorous, neutral phosphorous or
non-phosphorous linkages; and said sequence of linked units is
divided into at l ast two regions, wherein: a first of said regions
includes said .alpha.-nucleosides linked by charged and neutral
3'-5' phosphorous linkages, said .alpha.-nucleosides linked by
charged and neutral 2'-5' phosphorous linkages, said
.alpha.-nucleosides linked by non-phosphorous linkages, said
4'-thionucleosides linked by charged and neutral 3'-5' phosphorous
linkages, said 4'-thionucleosides linked by charged and neutral
2'-5' phosphorous linkages, said 4'-thionucleosides linked by
non-phosphorous linkages, said carbocyclic-nucleosides linked by
charged and neutral phosphorous linkag s, said
carbocyclic-nucleosides linked by non-phosphorous linkag s, said
.beta.-nucl sid s linked by charged and n utral 3'-5' linkages,
said .beta.-nucleosides linked by charged and neutral 2'-5'
linkages, and said .beta.-nucl osid s linked by non-phosphorous
linkages; and a second of said regions including said nucleobases
linked by non-phosphorous linkages and nucleobases that are
attached to phosphate linkages via a non-sugar tethering
moiety.
40. The macromolecule of claim 38 wherein said non-phosphate
linkage is a peptide linkage.
41. A macromolecule of claim 38 including a plurality of said first
regions.
42. A macromolecule of claim 38 including a plurality of said
second regions.
43. A macromolecule of claim 41 including a plurality of said first
regions.
44. A method of treating an organism having a disease characterized
by the undesired production of a protein comprising contacting the
organism with a compound of claim 34.
45. A pharmaceutical composition comprising a pharmaceutically
effective amount of a compound of claim 34 and a pharmaceutically
acceptable diluent or carrier.
46. A method of modifying in vitro a sequenc-specific nucleic acid,
comprising contacting a test solution containing a RNase H and said
nucleic acid with a compound f claim 34.
47. A method of treating an organism having a disease characterized
by the undesired production of a protein comprising contacting the
organism with a compound of claim 39.
48. A pharmaceutical composition comprising a pharmaceutically
effective amount of a compound of claim 39 and a pharmaceutically
acceptable diluent or carrier.
Description
FIELD OF THE INVENTION
[0001] This invention is directed to the synthesis and use of
oligonucleotides and macromolecules to elicit RNase H for strand
cleavage in an opposing strand. Included in the invention are
oligonucleotides wherein at least some of the nucleotides of the
oligonucleotides are functionalized to be nuclease resistant, at
least some of the nucleotides of the oligonucleotide include a
substituent that potentiates hybridization of the oligonucleotide
to a complementary strand, and at least some of the nucleotides of
the oligonucleotide include 2'-deoxy-erythro-pentofuranosyl sugar
moieties. The oligonucleotides and macromolecules are useful for
therapeutics, diagnostics and as research reagents.
BACKGROUND OF THE INVENTION
[0002] It is well known that most of the bodily states in mammals
including most disease states, are effected by proteins. Such
proteins, either acting directly or through their enzymatic
functions, contribute in major proportion to many diseases in
animals and man. Classical therapeutics has generally focused upon
interactions with such proteins in an effort to moderate their
disease causing or dis ase potentiating functions. Recently,
however, attempts have been made to moderate the actual production
of such proteins by interactions with messenger RNA (mRNA) or other
intracellular RNA's that direct protein synthesis. It is gen rally
the object of such therapeutic approaches to int rfere with r th
rwise modulate gene expression leading t und sired pr tein
formation.
[0003] Antisense method logy is the c mplementary hybridization of
relatively short oligonucleotides to single-stranded RNA or
single-stranded DNA such that the normal, essential functions of
these intracellular nucleic acids are disrupted. Hybridization is
the sequence specific hydrogen bonding via Watson-Crick base pairs
of the heterocyclic bases of oligonucleotides to RNA or DNA. Such
base pairs are said to be complementary to one another.
[0004] Naturally occurring events that provide for the disruption
of the nucleic acid function, as discussed by Cohen in
Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC
Press, Inc., Boca Raton, Fla. (1989) are thought to be of tw types.
The first is hybridization arrest. This denotes the terminating
event in which an oligonucleotide inhibitor binds to target nucleic
acid and thus prevents, by simple steric hindrance, the binding of
essential proteins, most often ribosomes, to the nucleic acid.
Methyl phosphonate oligonucleotides (see, e.g., Miller, et al.,
Anti-Cancer Drug Design 1987, 2, 117) and .alpha.-anomer
oligonucleotides are the tw most extensively studied antisense
agents that are thought t disrupt nucleic acid function by
hybridization arrest.
[0005] In determining the extent of hybridization arrest of an
oligonucleotide, the relative ability of an oligonucleotide to bind
to complementary nucleic acids may be compared by determining the
melting temperature of a particular hybridization complex. The
melting temperature (T.sub.m), a characteristic physical property
of double helixes, denotes the temperature in degrees centigrade at
which 50% helical (hybridized) versus coil (unhybridized) forms are
present. T.sub.m is measured by using the UV spectrum to determine
the formati n and breakdown (melting) of hybridization. Base
stacking which occurs during hybridization, is accompanied by a
reduction in UV absorption (hypochromicity). Consequently a
reduction in UV absorption indicates a higher T.sub.m. Th higher
the T.sub.m, the greater th strength of the binding of the strands.
Non-Watson-Crick base pairing, i.e. bas mismatch, has a str ng
destabilizing effect on the T.sub.m.
[0006] Th second type f terminating vent for antisense
oligonucleotides involves the enzymatic cleavage of the targeted
RNA by intracellular RNase H. The mechanism of such RNase H
cleavages requires that a 2'-deoxyribofuranosyl olig nucleotide
hybridize to a targeted RNA. The resulting DNA-RNA duplex activates
the RNase H enzyme; the activated enzyme cleaves the RNA strand.
Cleavage of the RNA strand destroys the normal function of the RNA.
Phosphorothioate olig-nucleotides are one prominent example of
antisense agents that operate by this type of terminating event.
For a DNA oligonucleotide to be useful for activation of RNase H,
the oligonucleotide must be reasonably stable to nucleases in order
to survive in a cell for a time sufficient for the RNase H
activation.
[0007] Several recent publications of Walder, et al. further
describe the interaction of RNase H and oligonucleotides. Of
particular interest are: (1) Dagle, et al., Nucleic Acids Research
1990, 18, 4751; (2) Dagle, et al., Antisense Research And
Development 1991, 1, 11; (3) Eder, et al., J. Biol. Chem. 1991,
266, 6472; and (4) Dagle, et al., Nucleic Acids Research 1991, 19,
1805. In these papers, Walder, et al. note that DNA
oligonucleotides having both unmodified phosphodiester
internucleoside linkages and modified, phosphorothi at
internucleoside linkages are substrates for cellular RNase H. Since
they are substrates, they activate the cleavage of target RNA by
the RNase H. However, the authors further note that in Xenopus
embryos, both phosphodiester linkages and phosph rothioate linkages
are also subject to exonuclease degradati n. Such nuclease
degradation is detrimental since it rapidly depletes the
oligonucleotide available for RNase H activation.
[0008] As described in references (1), (2), and (4), to stabilize
their oligonucleotides against nuclease degradation while still
providing for RNase H activation, Walder, t al. constructed
2'-deoxy oligonucl tides having a short secti n of phosphodiester
linked nucl tides p siti ned between sections f phosphoramidat ,
alkyl phosphonate or phosphotriest r linkages. While th ph
sphoamidate-containing oligonucleotid s were stabiliz d against x
nucleases, in reference (4) the authors noted that each
phosphoramidate linkage resulted in a loss of 1.6.degree. C. in the
measured T.sub.m value of the phosphoramidat containing
oligonucleotides. Such decrease in the T.sub.m value is indicative
of an undesirable decrease in the hybridization between the
oligonucleotide and its target strand.
[0009] Other authors have commented on the effect such a loss of
hybridization between an antisense oligonucleotide and its targeted
strand can have. Saison-Behmoaras, et al., EMBO Journal 1991, 10,
1111, observed that even through an oligonucleotide could be a
substrate for RNase H, cleavage efficiency by RNase H was low
because of weak hybridization to the mRNA. The authors also noted
that the inclusion of an acridin substitution at the 3' end of the
oligonucleotide protected th oligonucleotide from exonucleases.
[0010] While it has been recognized that cleavage of a target RNA
strand using an antisense oligonucleotide and RNase H would be
useful, nuclease resistance of the oligonucleotide and fidelity of
the hybridization are also of great importance. Heretofore, there
have been no suggestion in the art of methods or materials that
could both activate RNase H while concurrently maintaining or
improving hybridization properties and providing nuclease
resistance even though there has been a long felt need for such
methods and materials. Accordingly, there remains a long-felt need
for such methods and materials.
OBJECTS OF THE INVENTION
[0011] It is an object of this invention t pr vide oligonucleotides
that both activate RNase H upon hybridization with a target strand
and resist nuclease degradation.
[0012] It is a further object to provide oligonucleotides that
activate RNase H, inhibit nuclease degradation, and provide
improved binding affinity between the oligonucleotide and the
target strand.
[0013] A still further object is to provide research and diagnostic
methods and materials for assaying bodily stat s in animals,
especially diseased states.
[0014] Another object is to provide therapeutic and research
methods and materials for the treatment of diseases thr ugh
modulation of the activity of DNA and RNA.
BRIEF DESCRIPTION OF THE INVENTION
[0015] In accordance with one embodiment of this invention there
are provided oligonucleotides formed from a sequence of nucleotide
units. The oligonucleotides incorporate a least one nucleotide unit
that is functionalized to increase nuclease resistance of the
oligonucleotides. Further, at least some of the nucleotide units of
the oligonucleotides are functionalized with a substituent group to
increase binding affinity of the oligonucleotides to target RNAs,
and at least some of the nucleotide units have
2'-deoxy-erythro-pentofuranosyl sugar moieties.
[0016] In preferred oligonucleotides of the invention, nucleotide
units that are functionalized for increased binding affinity are
functionalized to include a 2'-substituent group. In even more
preferred embodiments, the 2'-substituent group is fluoro, C1-C9
alkoxy, C1-C9 aminoalkoxy including aminopropoxy, allyloxy,
C.sub.1-C.sub.9-alkyl-imidazole and polyethylene glycol. Preferred
alkoxy substituents include methoxy, ethoxy and propoxy. A
preferred aminoalkoxy unit is aminopropoxy. A preferred
alkyl-imidazol is 1-propyl-3-(imidazoyl).
[0017] In certain preferred ligonucle tides of the invention having
increased nucl as r sistanc , each nucl otide unit of the
oligonucleotides is a ph sphorothioat or phosphorodithioate nucl
otide. In ther preferred oligonucleotides, the 3' terminal
nucleotide unit is functionalized with either or both of a 2' or a
3' substituent.
[0018] The oligonucleotides include a plurality of nucleotide units
bearing substituent groups that increase binding affinity of the
oligonucleotide to a complementary strand of nucleic acid. In
certain preferred embodiments, the nucleotide units that bear such
substituents can be divided into a first nucleotide unit
sub-sequence and a second nucleotide unit sub-sequence, with
2'-deoxy-erythro-pentofuranosyl structures being positioned within
the oligonucleotide between the first nucleotide unit sub-sequence
and the second nucleotide unit sub-sequence. It is preferred that
all such intervening nucleotide units be
2'-deoxy-erythro-pentofuranosyl units.
[0019] In further preferred oligonucleotides of the invention,
nucleotide units bearing substituents that increase binding
affinity are located at one or both of the 3' or the 5' termini of
the oligonucleotide. There can be from one to about eight
nucleotide units that are substituted with substitu nt groups.
Preferably, at least five sequential nucleotide units are
2'-deoxy-erythro-pentofuranosyl sugar moieties.
[0020] The present invention also provides macromolecules formed
from a plurality of linked nucleosides selected from
.alpha.-nucleosides, .beta.-nucleosides including
2'-deoxy-erythro-pentofuranosyl .beta.-nucleosides,
4'-thionucleosides, and carbocyclic-nucleosides. These nucleosides
are connected by linkages in a sequence that is hybridizable to a
complementary nucleic acid. The linkages are selected from charged
phosphorous linkages, neutral phosphorous linkages, and
non-phosphorous linkages. The sequence of linked nucleosides is
divided into at least two regions. The first nucleoside region
includes the following types of nucleosides: .alpha.-nucleosides
linked by charged and neutral 3'-5' phosphorous linkages;
.alpha.-nucleosides linked by charged and neutral 2'-5' ph sphorous
linkages; .alpha.-nucle sid s linked by n n-ph sph rous linkages;
4'-thionudl osides linked by charg d and neutral 3'-5' phosphorous
linkages; 4'-thionucleosides link d by charged and neutral 2'-5'
phosphorous linkages; 4'-thionucleosides linked by non-phosphorous
linkages; carbocyclic-nucleosides linked by charged and neutral
3'-5' phosphorous linkages; carbocyclic-nucleosides linked by
charged and neutral 2'-5' phosphorous linkages;
carbocyclic-nucleosides linked by non-phosphorous linkages;
.beta.-nucleosides linked by charged and neutral 2'-5' linkages;
and .beta.-nucleosides linked by non-phosphorous linkages. A sec nd
nucleoside region consists of 2'-deoxy-erythro-pentofuranosyl
.beta.-nucleosides linked by charged 3'-5' phosphorous linkages
having negative charge at physiological pH. In preferred
embodiments, the macromolecules include at least 3 of said
2'-deoxy-erythro-pentofuranosyl .beta.-nucleosides, more preferably
at least 5 of said 2'-deoxy-erythro-pentofuranosyl
.beta.-nucleotides. In further preferred embodiments there exists a
third nucleoside region whose nucleosides are selected from those
selectable for the first region. In preferred embodiments th second
region is positioned between the first and third regions.
[0021] Preferred charged phosphorous linkages includ
phosphodiester, phosphorothioate, phosphorodithioate,
phosphoroselenate and phosphorodiselenate linkages; phosphodiester
and phosphorothioate linkages are particularly preferred. Preferred
neutral phosphorous linkages include alkyl and aryl phosphonates,
alkyl and aryl phosphoroamidites, alkyl and aryl phosphotriesters,
hydrogen phosphonat and boranophosphate linkages. Preferred
non-phosphorous linkages include peptide linkages, hydrazine
linkages, hydroxy-amine linkages, carbamate linkages, morpholine
linkages, carb nate linkages, amide linkages, oxymethyleneimine
linkages, hydrazide linkages, silyl linkages, sulfide linkages,
disulfide linkag s, sulfone linkages, sulfoxide linkages, sulfonate
linkag s, sulfonamide linkages, formacetal linkages, thioformacetal
linkages, oxime linkages and ethyl ne glycol linkages.
[0022] The inv ntion also provid s macromol cules formed from a
plurality f link d units, ach f which is selected from nucleosid s
and nucl obases. The nucleosides include .alpha.-nucleosid s,
.beta.-nucle sides including 2'-d oxy-erythro-pento-furanosyl
.beta.-nucleosides, 4'-thionucleosides and carbocyclic-nucleosides.
The nucleobases include purin-9-yl and pyrimidin-1-yl heterocyclic
bases. The nucleosides and nucleobases of the units are linked
together by linkages in a sequence wherein the sequence is
hybridizable to a complementary nucleic acid and the sequence of
linked units is divided into at least tw regions. The linkages are
selected from charged 3'-5' phosphorous, neutral 3'-5' phosphorous,
charged 2'-5' phosphorous, neutral 2'-5' phosphorous or
non-phosphorous linkages. A first of the regions includes
nucleobases linked by non-phosphorous linkages and nucleobases that
are attached to phosphate linkages via non-sugar tethering groups,
and nucleosides selected from .alpha.-nucleosides linked by charged
and neutral 3'-5' phosphorous linkages, .alpha.-nucleosides linked
by charged and neutral 2'-5' phosphorous linkages,
.alpha.-nucleosides linked by non-phosphorous linkages,
4'-thionucleosides linked by charg d and neutral 3'-5' phosphorous
linkages, 4'-thionucleosides linked by charged and neutral 2'-5'
phosphorous linkages, 4'-thionucleosides linked by non-phosphorous
linkages, carbocyclic-nucleosides linked by charged and neutral
3'-5' phosphorous linkages, carbocyclic-nucleosides linked by
charged and neutral 2'-5' phosphorous linkages,
carbocyclic-nucleosides linked by non-phosphorous linkages,
.beta.-nucleosides linked by charged and neutral 2'-5' linkages,
and .beta.-nucleosides linked by non-phosphorous linkages. A second
of the regions includ s only 2'-deoxy-erythro-pentofur- anosyl
.beta.-nucleosides linked by charged 3'-5' phosphorous linkages
wherein the 3'-5' phosphorous linkages have a negative charge at
physiological pH.
[0023] In certain preferred embodiments, the first regi n includes
at least two nucleobases joined by a non-phosphate linkage such as
a peptide linkage. In preferred embodiments, the macromolecules
include a third region that is selected from the same groups as
described above f r the first r gion. In preferred embodiments, the
sec nd regi n is located between th first and third r gions.
[0024] The inventi n also provides macromolecules that have a
plurality of linked units, each of which is selected from
nucleosides and nucleobases. The nucleosides are selected fr m
.beta.-nucleosides, .beta.-nucleosides, 4'-thionucleosides and
carb-cyclic-nucleosides and the nucleobases are selected from
purin-9-yl and pyrimidin-1-yl heterocyclic bases. The nucleosides
and nucleobases of said units are linked together by linkag s in a
sequence wherein the sequence is hybridizable to a complementary
nucleic acid. The sequence of linked units is divided into at least
two regions. The linkages are selected from charged phosphorous,
neutral phosphorous or non-phosphorous linkages. A first of the
regions include .alpha.-nucleosides linked by charged and neutral
3'-5' phosphorous linkages, .alpha.-nucleosides linked by charged
and neutral 2'-5' phosphorous linkages, .alpha.-nucleosides linked
by non-phosphorous linkages, 4'-thionucleosides linked by charged
and neutral 3'-5' phosphorous linkages, 4'-thionucleosides linked
by charged and neutral 2'-5' phosphorous linkages,
4'-thionucleosides linked by non-phosphorous linkages,
carbocyclic-nucleosides linked by charged and neutral phosphorous
linkages, carbocyclic-nucleosides linked by non-phosphorous
linkages, .beta.-nucleosides linked by charged and neutral 3'-5'
linkages, .beta.-nucleosides linked by charged and neutral 2'-5'
linkages, and .beta.-nucleosides linked by non-phosphorous
linkages. A second of the regions include nucleobases linked by
non-phosphorous linkages and nucleobases that are attached to
phosphate linkages via a non-sugar tethering moiety.
[0025] Preferred nucleobases of the invention include adenine,
guanine, cytosine, uracil, thymine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl and other alkyl adenines, 2-propyl and
other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil,
6-aza cytosine and 6-aza thymine, 5-uracil (pseudo uracil),
4-thiouracil, 8-halo adenine, 8-amino-adenine, 8-thiol adenine,
8-thiolalkyl adenines, 8-hydroxyl adenin and other 8 substituted
adenin s and 8-halo guanin s, 8-amino guanin, 8-thi l guanine,
8-thiolalkyl guanin s, 8-hydroxyl guanin and oth r 8 substituted
guanines, th r aza and d aza uracils, ther aza and deaza
thymidines, other aza and deaza cytosine, aza and deaza adenines,
aza and deaza guanines or 5-trifluoromethyl uracil and
5-trifluorocytosine.
[0026] The invention also provides methods of treating an organism
having a disease characterized by the undesir d production of an
protein. These methods include contacting the organism with an
oligonucleotide having a sequence of nucleotides capable of
specifically hybridizing to a complementary strand of nucleic acid
where at least one of the nucleotides is functionalized to increase
nuclease resistance of the oligonucleotide to nucleases, where a
substituent group located thereon to increase binding affinity of
th oligonucleotide to the complementary strand of nucleic acid and
where a plurality of the nucleotides have
2'-deoxy-erythroregions;-pentofuranosyl sugar moieties.
[0027] Further in accordance with this invention there are provided
compositions including a pharmaceutically effective amount of an
oligonucleotide having a sequence of nucleotides capable of
specifically hybridizing to a complementary strand of nucleic acid
and where at least one of the nucleotides is functionalized to
increase nuclease resistance of the olig nucleotide to nucleases
and where a plurality of the nucleotides have a substituent group
located thereon to increase binding affinity of the oligonucleotide
to th complementary strand of nucleic acid and where a plurality of
the nucleotides have 2'-deoxy-erythro-pentofuranosyl sugar
moieties. The composition further include a pharmaceutically
acceptable diluent or carrier.
[0028] Further in accordance with this invention there are provided
methods for in vitro modification of a sequence specific nucleic
acid including contacting a test solution containing an RNase H
enzyme and said nucleic acid with an oligonucleotid having a
sequence of nucl otides capable of specifically hybridizing to a c
mplementary strand of nucleic acid and wher at l ast ne f the nucl
otid s is functionalized to incr as nucl ase resistance of the olig
nucle tid to nucleases and where a plurality f the nucleotides hav
a substituent group located ther n to increase binding affinity of
the oligonucleotide to the complementary strand of nucleic acid and
where a plurality f the nucleotides have
2'-deoxy-erythro-pentofuranosyl sugar moieties.
[0029] There are also provided methods of concurrently enhancing
hybridization and RNase H enzyme activation in an organism that
includes contacting the organism with an oligonucleotide having a
sequence of nucleotides capable of specifically hybridizing to a
complementary strand of nucleic acid and where at least one of the
nucleotides is functionalized to increase nuclease resistance of
the oligonucleotide to nucleases and where a plurality of the
nucleotides have a substituent group located thereon t increase
binding affinity of the oligonucleotide to the complementary strand
of nucleic acid and where a plurality of the nucleotides have
2'-deoxy-erythro-pentofuranosyl sugar moieties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] This invention will be better understood when taken in
conjunction with the drawings wherein:
[0031] FIG. 1 is a graph showing dose response activity of
oligonucleotides of the invention and a reference compound; and
[0032] FIG. 2 is a bar chart showing dose response activity of
oligonucleotides of the invention and reference compounds.
DETAILED DESCRIPTION OF THE INVENTION
[0033] In accordance with the objects of this inventi n, novel
oligonucleotides and macromolecules that, at once, have increased
nuclease resistance, increased binding affinity to complementary
strands and that are substrates for RNase H are provided. The
oligonucleotides and macromolecules of the invention are assembled
fr m a plurality of nucleotide, nucleoside or nucleobas sub-units.
Each oligonucleotide or macromolecule of th invention includes at l
ast one nucle tide, nucle side r nucle base unit that is
functionalized to increase the nuclease resistances of the
oligonucleotide. Further, in certain embodiments of the invention
at least some of the nucleotide or nucleoside units bear a
substituent group that increases the binding affinity of the
oligonucleotide or macromolecule to a complementary strand of
nucleic acid. Additionally at least some of the nucleotide units
comprise a 2'-deoxy-erythro-pentofuranosyl group as th ir sugar
moiety.
[0034] In conjunction with the above guidelines, each nucleotide
unit of an oligonucleotides of the invention, alternatively
referred to as a subunit, can be a "natural" or a "synthetic"
moiety. Thus, in the context of this invention, the term
"oligonucleotide" in a first instance refers to a polynucleotide
formed from a plurality of joined nucleotide units. The nucleotides
units are joined together via native internucleoside,
phosphodiester linkages. The nucleotide units are formed from
naturally-occurring bases and pentofuran syl sugars groups. The
term "oligonucleotide" thus effectively includes naturally
occurring species or synthetic species formed from naturally
occurring nucleotide units.
[0035] Oligonucleotides of the invention also can include modified
subunits. The modifications can occur on the base portion of a
nucleotide, on the sugar portion of a nucleotide or on the linkage
joining one nucleotide to the next. In addition, nucleoside units
can be joined via connecting groups that substitute for the
inter-nucleoside phosphate linkages. Macromolecules of the type
have been identified as oligonucleosides. In such oligonucleosides
the linkages include an --O--CH.sub.2--CH.sub.2--O-- linkage (i.e.,
an ethylene glycol linkage) as well as other novel linkages
disclosed in the following U.S. patent application Ser. No.
566,836, filed Aug. 13, 1990, entitled Novel Nucleoside Analogs;
Ser. No. 703,619, filed May 21, 1991, entitl d Backbone Modifi d
Olig nucleotide Analogs; and Ser. No. 903,160, filed Jun. 24, 1992,
entitled Heteroatomic Oligonucl otide Linkag . Other m difications
can be made to the sugar, to th base, or to the phosphate gr up of
the nucleotid . R pr sentative modifications are discl sed in th
following U.S. patent application Ser. No. 463,358, filed Jan. 11,
1990, entitled Compositions And Methods For Detecting And
Modulating RNA Activity; Ser. No. 566,977, filed Aug. 13, 1990,
entitled Sugar Modified oligonucleotides That Detect And Modulate
Gene Expression; Ser. No. 558,663, filed Jul. 27, 1990, entitled
Novel Polyamine Conjugated Oligonucleotides; Ser. No. 558,806,
filed Jul. 27, 1991, entitled Nuclease Resistant Pyrimidine
Modified oligonucleotides That Detect And Modulate Gen Expression;
and Ser. No. PCT/US91/00243, filed Jan. 11, 1991, entitled
Compositions and Methods For Detecting And Modulating RNA Activity,
all assigned to the assignee of this invention. The disclosures of
each of the above noted patent applications are herein incorporated
by reference.
[0036] Thus, the terms oligonucleotide is intended to include
naturally occurring structures as well as non-naturally occurring
or "modified" structures--including modified sugar moieties,
modified base moieties or modified sugar linking moieties--that
function similarly to natural bases, natural sugars and natural
phosphodiester linkages. Thus, oligonucl otides can have altered
base moieties, altered sugar moieties or altered inter-sugar
linkages. Exemplary among these ar phosphorothioate,
phosphorodithioate, methyl phosphonate, phosphotriester,
phosphoramidate, phosphoroselenate and phosphorodiselenate
inter-nucleoside linkages used in place of phosphodiester
inter-nucleoside linkages; deaza or aza purines and pyrimidines
used in place of natural purine and pyrimidine bases; pyrimidine
bases having substituent groups at the 5 or 6 position; purine
bases having altered or replacement substituent groups at the 2, 6
or 8 positions; or sugars having substituent groups at their 2'
position, substitutions for one or more of the hydrogen atoms of
the sugar, or carbocyclic or acyclic sugar analogs. Th y may als c
mprise other modif icati ns c nsistent with the spirit f this
invention. Such oligonucl otides are best d scribed as being
functionally interchangeabl with natural oligonucleotides ( r
synthesized ligonucleotides along natural lines), but which have
one or more differences from natural structure. All such olig
nucleotides are comprehended by this invention so long as they
function effectively to mimic the structure of a desired RNA rDNA
strand.
[0037] In one preferred embodiment of this invention, nuclease
resistance is achieved by utilizing phosphorothioat internucleoside
linkages. Contrary to the reports of Wald r, et al. note above, I
have found that in systems such as fetal calf serum containing a
variety of 3'-exonucleases, modification of the internucleoside
linkage from a phosph -diester linkage to a phosphorothioate
linkage provides nuclease resistance.
[0038] Brill, at al., J. Am. Chem. Soc. 1991, 113, 3972, recently
reported that phosphorodithioate oligonucleotides also exhibit
nuclease resistance. These authors also reported that
phosphorodithioate oligonucleotide bind with complementary
deoxyoligonucleotides, stimulate RNase H and stimulate th binding
of lac repressor and cro repressor. In view of th se properties,
phosphorodithioates linkages also may be useful t increase nuclease
resistance of oligonucleotides of the invention.
[0039] Nuclease resistance further can be achieved by locating a
group at the 3' terminus of the oligonucleotide utilizing the
methods of Saison-Behmoraras, et al., supra, wherein a dodecanol
group is attached to the 3' terminus of the oligonucleotide. Other
suitable groups for providing increased nuclease resistance may
include steroid molecules and other lipids, reporter molecules,
conjugates and non-aromatic lipophilic molecules including
alicyclic hydrocarbons, saturated and unsaturated fatty acids,
waxes, terpenes and polyalicyclic hydrocarbons including adamantane
and buckminsterfullerenes. Particularly useful as steroid molecules
for this purpose are the bile acids including cholic acid,
deoxycholic acid and dehydrocholic acid. Other steroids include
cortisone, digoxigenin, test st rone and cholesterol and even
cationic steroids such as c rtis ne having a trimethylaminom thyl
hydrazide group attached via a doubl bond at the 3 position of the
cortisone ring. Particularly useful reporter molecules are biotin
and fluorescein dyes. Such groups can be attached to the 2'
hydroxyl group or 3' hydr xyl group of the 3' terminal nucleotide
either directly or utilizing an appropriate connector in the manner
described in U.S. patent application Ser. No. 782,374, filed Oct.
24, 1991 entitled Derivatized Oligonucleotides Having Improved
Uptake and Other Properties, assigned to the assignee as this
application, the entire contents of which are herein incorporated
by reference.
[0040] Attachment of functional groups at the 5' terminus of
compounds of the invention also may contribute to nuclease
resistance. Such groups include acridine groups (which also serves
as an intercalator) or other groups that exhibit either beneficial
pharmacokinetic or pharmacodynamic properties. Groups that exhibit
pharmacodynamic properties, in the context of this invention,
include groups that improve oligonucleotide uptake, enhance
oligonucleotide resistance to degradation, and/or strengthened
sequence-specific hybridization with RNA. Groups that exhibit
pharmacokinetic properties, in the context of this invention,
include groups that improve oligonucleotide uptake, distribution,
metabolism or excretion.
[0041] Further nuclease resistance is expect to be conferred
utilizing linkages such as the above identified
--O--CH.sub.2--CH.sub.2--O-- linkage and similar linkages of the
above identified U.S. patent applications Ser. No. 566,836, Ser.
No. 703,619, and Ser. No. 903,160, since these types f linkages do
not utilize natural phosphate ester-containing backbones that are
the natural substrates for nucleases. When nuclease resistance is
conferred upon an oligonucleotide of the invention by the use of a
phosphorothioate or other nuclease resistant internucleotide
linkages, such linkages will reside in each internucleotide sites.
In oth r embodiments, less than all f the internucl otide linkages
will be m dified to phosphorothioate or other nucl ase resistant
linkages.
[0042] I have found that binding affinity of olig nucleotides f the
invention can be increased by locating substituent groups on
nucleotide subunits of the oligonucleotides of the invention.
Preferred substituent groups are 2' substituent groups, i.e.,
substituent groups located at the 2' position of the sugar moiety
of the nucleotide subunits of the oligonucle tides of the
invention. Presently preferred substituent gr ups include but are
not limited to 2'-fluoro, 2'-alkoxy, 2'-amin -alkoxy, 2'-allyloxy,
2'-imidazole-alkoxy and 2'-poly(ethylene oxide). Alkoxy and
aminoalkoxy groups generally include lower alkyl groups,
particularly C1-C9 alkyl. Poly(ethylene glycols) are of the
structure (O--CH.sub.2--CH.sub.2).sub.n--O-alkyl. Particularly
preferred substituent groups are 2'-fluoro, 2'-methoxy, 2'-ethoxy,
2'-propoxy, 2'-aminopropoxy, 2'-imidazolepropoxy,
2'-imidazolebutoxy, and 2'-allyloxy groups.
[0043] Binding affinity also can be increased by the use of certain
modified bases in the nucleotide units that make up the
oligonucleotides of the invention. Such modified bases may include
6-azapyrimidines and N-2, N-6 and O-6 substitut d purines including
2-aminopropyladenine. Other modified pyrimidine and purine base are
expected to increase the binding affinity of oligonucleotides to a
complementary strand of nucleic acid.
[0044] The use of 2'-substituent groups increases the binding
affinity of the substituted oligonucleotides of the inventi n. In a
published study, Kawasaki and Cook, et al., Synthesis and
Biophysical Studies of 2'-dRIBO-F Modified Oligonucleotides,
Conference On Nucleic Acid Therapeutics, Clearwater, Fla., Jan.13,
1991, the inventor has reported a binding affinity increase of
1.6.degree. C. per substituted nucleotide unit of the
oligonucleotide. This is compared to an unsubstituted
oligonucleotide for a 15 mer phosphodiester oligonucleotide having
2'-deoxy-2'-fluoro groups as a substituent group on five of the
nucl otides of the oligonucle tide. When 11 of th nucleotides of
the oligonucl otid bor such 2'-deoxy-2'-fluoro substituent groups,
the binding affinity incr as d t 1.8.degree. C. p r substituted
nucl otide unit.
[0045] In that sam study, the 15 mer ph sphodiester oligonucleotide
was derivatized to th corresponding phosphorothioate analog. When
the 15 mer phosphodiester oligonucleotide was compared to its
phosphorothioate analog, the phosphorothioate analog had a binding
affinity of only about 66% of that of the 15 mer phosphodiester
oligonucleotide. Stated otherwise, binding affinity was lost in
derivatizing the oligonucleotide to its phosphorothioate analog.
However, when 2'-deoxy-2'-fluoro substituents were located at 11 of
the nucleotides of the 15 mer phosphorothioate oligonucleotide, the
binding affinity of the 2'-substituent groups more than overcame
the decrease noted by derivatizing the 15 mer oligonucleotide to
its phosphorothioate analog. In this compound, i.e., a 15 mer
phosphorothioate oligonucleotide having 11 nucleotide substituted
with 2'-fluoro groups, the binding affinity was increased to
2.5.degree. C. per substituent group. In this study no attempt was
made to include an appropriate consecutive sequence of nucleotides
have 2'-deoxy-erythro-pentofuranosyl sugars that would elicit RNase
H enzyme cleavage of a RNA target complementary to the
oligonucleotide of the study.
[0046] In order to elicit RNase H enzyme cleavage of a target RNA,
an oligonucleotide of the invention must include a segment or
sub-sequence therein that is a DNA type segment. Stated otherwise,
at least some of the nucleotide subunits of the oligonucleotides of
the invention must have 2'-deoxy-erythro-pentofuranosyl sugar
moieties. I have found that a sub-sequence having more than three
consecutive, linked 2'-de xy-erythro-pentofuranosyl-containing
nucleotide sub-units likely is necessary in order to elicit RNase H
activity upon hybridization of an oligonucleotide of the invention
with a target RNA. It is presently preferred to have a sub-sequence
of 5 or more consecutive 2'-deoxy-erythro-pentofuranosyl containing
nucleotide subunits in an oligonucleotide of the invention. Use of
at least 7 consecutive 2'-deoxy-erythro-pentofuranosyl-containing
nucl otide subunits is particularly pref rred.
[0047] Th mechanism of action of RNase H is recognition of a
DNA-RNA duplex followed by cl avage of the RNA stand of this
duplex. As noted in the Backgr und secti n above, others in the art
hav used modifi d DNA strands t impart nuclease stability to the
DNA strand. To do this they have used modified phosphate linkages
impart increased nuclease stability but detract from hybridization
properties. While I do not wish to be bound by theory, I have
identified certain nucleosides or nucleoside analogs that will
impart nuclease stability to an oligonucleotide, oligonucleoside or
other macromolecule and in certain instances also impart increase
binding to a complementary strand. These include
.alpha.-nucleosides linked by charged and neutral 3'-5' phosphorous
linkages, .alpha.-nucleosides linked by charged and neutral 2'-5'
phosphorous linkages, .alpha.-nucleosides linked by non-phosphorous
linkages, 4'-thionucleosides linked by charged and neutral 3'-5'
phosphorous linkages, 4'-thionucleosides linked by charged and
neutral 2'-5' phosphorous linkages, 4'-thionucleosides linked by n
n-phosphorous linkages, carbocyclic-nucleosides linked by charged
and neutral phosphorous linkages, carbocyclic-nucleosides linked by
non-phosphorous linkages, .beta.-nucleosides linked by charged and
neutral 3'-5' linkages, .beta.-nucleosides linked by charged and
neutral 2'-5' linkages, and .beta.-nucleosides linked by
non-phosphorous linkages. They further include nucleobases that are
attached to phosphate linkages via non-sugar tethering groups or
are attached to non-phosphate linkages.
[0048] Again, while not wishing to be bound by any particular
theory, I have found certain criteria that must be met f r RNase H
to recognize and elicit cleavage of a RNA strand. The first of
these is that the RNA stand at the cleavage site must have its
nucleosides connected via a phosphate linkage that bears a negative
charge. Additionally, the sugar of the nucleosides at the cleavage
site must be a .beta.-pentofuranosyl sugar and also must be in a 2'
endo conformation. The only nucleosides (nucleotides) that fit this
criteria are phosphodiester, phosphorothioate, phosphorodithioat ,
phosphoros l nate and phosphorodisel nat nucleotides of 2'-d
oxy-erythro-pentofuranosyl .beta.-nucleosid s.
[0049] In view of the above criteria, even certain nucleosides that
have been sh wn to reside in a 2' endo conformation ( .g., cyclop
ntyl nucleosides) will not elicit RNase H activity since they do
not incorporate a pentofuranosyl sugar. Modeling has shown that
oligonucleotide 4'-thionucleosides also will not elicit RNase H
activity, even though such nucleosides reside in an envelope
conformation, since they do not reside in a 2' endo conformati n.
Additionally, since .alpha.-nucleosides are of the opposite
configuration from .beta.-pentofuranosyl sugars they also will not
elicit RNase H activity.
[0050] Nucleobases that are attached to phosphate linkages via
non-sugar tethering groups or via non-phosphate linkages also do
not meet the criteria of having a .beta.-pentofuranosyl sugar in a
2' endo conformation. Thus, they likely will not elicit RNase H
activity.
[0051] As used herein, .alpha. and .beta. nucleosides include
ribofuranosyl, deoxyribofuranosyl (2'-deoxy-erythro-pentofuranosyl)
and arabinofuranosyl nucleosides. 4'-Thionucleosides are
nucleosides wherein the 4' ring oxygen atom of the pentofuranosyl
ring is substituted by a sulfur atom. Carbocyclic nucleosides are
nucleosides wherein the ring oxygen is substituted by a carbon
atom. Carbocyclic nucleosides include cyclopropyl, cyclobutyl,
cyclopentyl and cyclohexyl rings (C.sub.3-C.sub.6-carbocyclic)
having an appropriate nucleobase attached thereto. The above
.alpha. and .beta. nucleosides, 4'-thionucleosides and carbocyclic
nucleosides can include additional functional groups on their
heterocyclic base moiety and additional functional groups on those
carbon atoms of sugar or carbocyclic moiety that are not utilized
in linking the nucleoside in a macromolecule of the invention. For
example, substituent groups can be placed on the 1, 2, 3, 6, 7 or 8
position of purine heterocycles, the 2, 3, 4, 5 or 6 position of
pyrimidine heterocycles. Deaza and aza analogs of the purine and
pyrimidine heterocycles can be selected or 2' substituted sugar
derivatives can be selected. All f these types of substitutions ar
known in the nucleoside art.
[0052] .alpha.-Nucl osides hav been incorporat d into oligo-nucle
tides; as reported by Gagn r, t. al., Nucl ic Acids Res arch 1987,
15, 10419, they do not support RNase H degradation. Carbocyclic
modified oligonucleotides have been synthesized by a number of
investigators, including Perbost, et al., Biochemical and
Biophysical Research Communications 1989, 165, 742; Sagi, et al.,
Nucleic Acids Research 1990, 18, 2133; and Szemzo, et. al.,
Tetrahedron Letters 1990, 31, 1463. 4'-Thionucleosides have been
known for at least 25 years. An improved synthesis via
4'-thioribofuranose recently was reported by Secrist, et. al.,
Tenth International Roundtabl : Nucleosides, Nucleotides and Their
Biological Evaluation, Sep. 16-20, 1992, Abstracts of Papers,
Abstract 21 and in published patent application PCT/US91/02732.
[0053] For incorporation into oligonucleotides or oligonucleotide
suggorates, .alpha. and .beta. nucleosides, 4'-thionucleosides and
carbocyclic nucleosides will be blocked in the 5' positi n (or the
equivalent to the 5' position for the carbocyclic nucleosides) with
a dimethoxytrityl group, followed by phosphitylation in the 3'
position as per the tritylation and phosphitylation procedures
reported in Oligonucleotides and Analogs, A Practical Approach,
Eckstein, F., Ed.; The Practical Approach Series, IRL Press, New
York, 1991. Incorporation into oligonucleotides will be
accomplished utilizing a DNA synthesizer such as an ABI 380 B model
synthesizer using appropriate chemistry for the formation of
phosphodiester, phosphorothioate, phosphorodithioate or
methylphosphonates as per the synthetic protocols illustrated in
Eckstein op. cit.
[0054] Boranophosphate linked oligonucleotides are prepared as per
the methods described in published patent application PCT/US/06949.
Phosphoroselenates and phosphorodiselenates linked oligonucleotides
are prepared in a manner analogous t their thio counterparts using
the reagent 3H-1,2-benzothia-seleno-3-ol for introducing the seleno
moiety. This reagent is also useful for preparing
selenothio-phosphates from corr sponding H-phosphonothiate di ster
as report d by Stawinski, t al. T nth Int rnational Roundtabl :
Nucl osid s, Nucl otides and Th ir Biological Evaluation, Sep.
16-20, 1992, Abstracts of Papers, Abstract 80. Hydrogen
phosphonate-linked oligonucleotides--as well as alkyl and aryl
phosphonate, alkyl and aryl phosphotriesters and alkyl and aryl
phosphoramidates linked oligonucleotides--are prepared in the mann
r of published patent application PCT/US88/03842. This patent
application also discusses the preparation of phosphorothioat s and
phosphoroselenates linked oligonucleotides.
[0055] Non-phosphate backbones include carbonate, carbamat silyl,
sulfide, sulfone, sulfoxide, sulfonate, sulfonamide, formacetal,
thioformacetal, oxime, hydroxylamine, hydrazine, hydrazide,
disulfide, amide, urea and peptide linkages. Oligonucleoside having
their nucleosides connected by carbonat linkages are prepared as
described by, for example, Mertes, t al., J. Med. Chem. 1969, 12,
154 and later by others. Oligonucleoside having their nucleosides
connected by carbamate linkages are prepared as was first described
by Gait, et. al., J. Chem. Soc. Perkin 1 1974, 1684 and later by
others. Oligonucleoside having their nucleosides connect by silyl
linkages are prepared as described Ogilvie, et al., Tetrahedron
Letters 1985, 26, 4159 and Nucleic Acids Res. 1988, 16, 4583.
Oligonucleoside having their nucleosides connected by sulfid
linkages and the associated sulfoxide and sulfone linkages ar
prepared as described by Schneider, et al., Tetrahedron Letters
1990, 31, 335 and in other publications such as published patent
application PCT/US89/02323.
[0056] Oligonucleoside having their nucleosides connected by
sulfonate linkages are prepared as described by Musicki, t al.,
Org. Chem. 1991, 55, 4231 and Tetrahedron Letters 1991, 32, 2385.
Oligonucleoside having their nucleosides connect d by sulfonamide
linkages are prepared as described by Kirshenbaum, et. al., The 5th
San Diego Conference: Nucleic Acids: New Frontiers, Poster abstract
28, Nov. 14-16, 1990. Oligonucleoside having their nucleosides
connected by formacetals are prepared as described by Matteucci, T
trah dron Lett rs 1990, 31, 2385 and V en man, t. al., R cu il d s
Trav. Chim. 1990, 109, 449 as well as by the pr cedures of
published patent application PCT/US90/06110. Olig nucle side having
their nucleosides connected by thioformacetals are prepared as
described by Matteucci, et. al., J. Am. Chem. Soc. 1991, 113, 7767;
Matteucci, Nucleosides & Nucleotides 1991, 10, 231, and the
above noted patent application PCT/US90/06110.
[0057] Oligonucleoside having their nucleosides connected by oxime,
hydroxylamine, hydrazine and amide linkages will be prepared as per
the disclosures of U.S. patent application Ser. No. 703,619 filed
May 21, 1991 and related PCT patent applications PCT/US92/04292 and
PCT/US92/04305 as well as corresponding published procedures by
myself and co-authors in Vasseur, et. al., J. Am. Chem. Soc. 1992,
114, 4006 and Debart, et. al., Tetrahedron Letters 1992, 33, 2645.
Oligonucleoside having their nucleosides connect by morpholine
linkages will be prepared as described in U.S. Pat. No.
5,034,506.
[0058] Further non-phosphate linkage suitable for use in this
invention include linkages have two adjacent heteroatoms in
combination with one or two methylene moieties. Oligonucle sides
having their nucleosides connect by such linkages will be prepared
as per the disclosures of U.S. patent application Ser. No. 903,160,
filed Jun. 24, 1992, the entire disclosure of which is herein
incorporated by reference.
[0059] Structural units having nucleobases attached via
non-phosphate linkages wherein the non-phosphate linkages are
peptide linkages will be prepared as per the procedures f patent
application PCT/EP/01219. For use in preparing such structural
units, suitable nucleobase include adenine, guanin , cytosine,
uracil, thymine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl
and other alkyl derivatives of adenine and guanine, 2-propyl and
other alkyl derivatives of adenine and guanine, 5-halo uracil and
cytosine, 6-azo uracil, cytosin and thymine, 5-uracil (pseudo
uracil), 4-thiouracil, 8-hal , amino, thiol, thiolalkyl, hydroxyl
and other 8 substituted ad nines and guanines, 5-trifluorom thyl
and other 5 substituted uracils and cytosin s, 7-methylguanine and
ther nucle bas such as those disclosed in U.S. Pat. No.
3,687,808.
[0060] Peptide linkages include 5, 6 and 7 atom l ng backbones
connected by amide links. Other, similar n n-phosphate backbones
having ester, amide and hydrazide links are prepared as per
published patent applications PCT/US86/00544 and
PCT/US86/00545.
[0061] Other .alpha. and .beta. nucleosides, 4'-thionucleoside and
carbocyclic nucleosides having the heturocyclic bases as disclosed
for the nucleobases above can be prepared and incorporated in to
the respective .alpha. and .beta. nucleosides, 4'-thionucleoside
and carbocyclic nucleosides.
[0062] Non-sugar tethering groups include 3,4-dihydroxybutyl (see,
Augustyns, et. al., Nucleic Acids Research 1991, 19, 2587) and
dihydroxyproproxymethyl (see, Schneider, et al., J. Am. Chem. Soc.
1990, 112, 453) and other linear chains such as C.sub.1-C.sub.10
alkyl, alkenyl and alkynyl. While the 3,4-dihydroxybutyl and
dihydroxyproproxymethyl non-sugar tethering groups are the acyclic
fragments of a .beta.-pentofuranosyl sugar, they will not serve to
elicit RNase H activation. Preferred for a non-sugar tethering
groups is the 3,4-dihydroxybutyl groups since the
dihydroxyproproxymethyl when used in an oligonucleotide analog upon
hybridization has shown a suppression of the melting temperature
between it and a complementary nucleic strand.
[0063] Normal 3'-5' phosphodiester linkages of natural nucleic
acids have 3 hetero atoms (--O--P--O--) between the respective
sugars of the adjacent nucleosides. If the 5' methylene group (the
5' CH.sub.2 group of the 3' nucleoside of th adjacent nucleosides)
is also included, these phosphodiester linked nucleic acids can be
viewed as being connected via linkages that are 4 atoms long.
[0064] Two strands of .beta.-oligonucleotides will hybridize with
each other with an anti-parallel polarity while a strand of
.alpha.-oligonucleotides will hybridize with strand of
.beta.-oligonucleotides with a parallel polarity. In c rtain
embodiments, oligonucleotides of the inventi n will hav a r gi n
formed of .alpha.-nucleotides and a further region f rmed of
.beta.-nucl otid s. These two regions are c nnected via an
inter-regi n linkage. For such an olig nucle tide to bind t a c
rresponding complementary .beta. strand of a nucleic acid and
maintain th parallel polarity of the .alpha. region simultaneously
with the anti-parallel polarity of the .beta. region, either a
3'-3' connection or a 5'-5' connection must be made between the
.alpha. and .beta. regions f the oligonucleotide of the invention.
The 3'-3' connection (having no 5' methylene moieties) yields a 3
atom long linkage, while the 5'-5' connection (having two 5'
methylene moieties) yields a 5 atom long linkage.
[0065] For embodiments of the invention wherein a 4 atom l ng
linkage between adjacent .alpha. and .beta. regions is desired, use
of a symmetrical linking nucleoside or nucleoside surrogate will
yield a 4 atom long linkage between each adjacent nucleoside pair.
An example of such a symmetrical linking nucleoside surrogate is a
3,3-bis-hydroxylmethyl cyclobutyl nucleoside as disclosed in my
U.S. patent application Ser. No. 808,201, filed Dec. 13, 1991,
entitled Cyclobutyl Oligonucleotide Surrogates, the entire
disclosure of which is herein incorporated by reference.
[0066] Other suitable linkages to achieve 4 atom spacing will
include alicyclic compounds of the class
1-hydroxyl-2-hydroxyl-methyl-alk-.omega.- -yl type moieties wherein
a nucleobase is connected to the .omega. (omega or last) position.
Examples of this type f linkage are
9-(1-hydroxyl-2-methylhydroxyl-pent-5-yl)adenine,
9-(1-hydroxyl-2-methylh- ydroxyl-pent-5-yl)guanine,
1-(1-hydroxyl-2-methylhydroxyl-pent-5-yl)uridin- e,
1-(1-hydroxyl-2-methylhydroxyl-pent-5-yl)cytosine and the
corresponding 3, 4 and 7 atom analogs, wherein a propyl, butyl or
hexyl alkyl group is utilized in place of the pentyl group. A
further example includes a nucleoside having a pentofuranosyl sugar
that is substituted with a 4'-hydroxylmethy group. In this instance
the linkages to the 5' nucleoside is an normal linkage via the
normal 5' hydroxyl moiety, whereas the linkage to the 3' nucleoside
is not through th normal 3'-hydroxyl group but is through the
4'-hydroxylmethy moiety. As with the cycl butyl nucleoside, with
both the alicyclic moieties r the 4'-substitut d nucleoside m
ieties, a 4 at m long linkage is achieved betw en adjacent regions
of the oligonucleotide f the invention.
[0067] In a manner similar to that described above, in th se
embodiments of this invention that have adjacent regions of a
macromolecule formed from different types of moieties, an
interconnection of a desired length can be formed between each of
the two adjacent regions of the macromolecule. The symmetrical
interconnection is achieved by selecting a linking moiety that can
form a covalent bond to both of the different types of moieties
forming the adjacent regions. The linking moiety is selected such
that the resulting chain of atoms between the linking moiety and
the different types of moieties is of the same length.
[0068] The oligonucleotides and macromolecules of th invention
preferably comprise from about 10 to about 30 nucleotide or
nucleobase subunits. It is more preferred that such
oligonucleotides and macromolecules comprise from about 15 to about
25 subunits. As will be appreciated, a subunit is a base and sugar
combination suitably bound to adjacent subunits through
phosphorothioate or other linkages or a nucleobase and appropriate
tether suitable bound to adjacent subunits through phosphorous or
non-phosphorous linkages. Such terms are us d interchangeably with
the term "unit." In order to elicit a RNase H response, as
specified above, within this total overall sequence length of the
oligonucleotide or macromolecule will b a sub-sequence of greater
than 3 but preferably five or more consecutive
2'-deoxy-erythro-pentofuranosyl containing nucleotide subunits.
[0069] It is presently preferred to incorporated the
2'-deoxy-erythro-pentofuranosyl-containing nucleotide sub-sequ nce
within the oligonucleotide or macromolecule main sequence such that
within the oligonucleotide or macromolecule ther nucleotide
subunits of the oligonucleotide or macromolecul are located on
either sid of the 2'-deoxy-erythro-pentofuranosyl nucleotide
sub-sequence.
[0070] In certain emb dim nts of the invention, if the remainder f
the nucleotide subunits each include a 2'-substituent group f r
increased binding affinity, then the
2'-deoxy-erythro-pentofuranosyl nucleotide sub-sequence will be
located between a first sub-sequence of nucleotide subunits having
2'-substituent groups and a second sub-sequence of nucleotide
subunits having 2'-substituent groups. Other constructions are also
possible, including locating the 2'-deoxy-erythro-pentofuranosyl
nucleotide sub-sequence at either the 3' or the 5' terminus of the
oligonucleotide of the invention.
[0071] Compounds of the invention can be utilized in diagnostics,
therapeutics and as research reagents and kits. They can be
utilized in pharmaceutical compositions by including an effective
amount of oligonucleotide of the invention admixed with a suitable
pharmaceutically acceptable diluent or carrier. They further can be
used for treating organisms having a disease characterized by the
undesired production of a protein. The organism can be contacted
with an oligonucleotide of the invention having a sequence that is
capable of specifically hybridizing with a strand of nucleic acid
that codes for the undesirable protein.
[0072] Such therapeutic treatment can be practiced in a variety of
organisms ranging from unicellular prokaryotic and eukaryotic
organisms to multicellular eukaryotic organisms. Any organism that
utilizes DNA-RNA transcription or RNA-protein translation as a
fundamental part of its hereditary, metab lic or cellular control
is susceptible to such therapeutic and/or prophylactic treatment.
Seemingly diverse organisms such as bacteria, yeast, protozoa,
algae, all plant and all high r animal forms, including
warm-blooded animals, can be treated by this therapy. Further,
since each of the cells of multicellular eukaryotes also includes
both DNA-RNA transcription and RNA-protein translation as an
integral part of their cellular activity, such therapeutics and/or
diagnostics can also be practiced on such cellular populations.
Furthermore, many f th rganelles, .g., mitochondria and
chloroplasts, f eukaryotic cells also include transcription and
translation mechanisms. As such, single cells, cellular p pulations
or organ lles also can be included within the definition of
organisms that are capable of being treated with the therapeutic or
diagnostic oligonucleotides of the invention. As used herein,
therapeutics is meant to include both the eradication of a disease
state, killing of an organism, e g., bacterial, protozoan or other
infection, r control of erratic or harmful cellular growth or
expressi n.
[0073] For purpose of illustration, the compounds of the invention
have been used in a ras-luciferase fusion system using
ras-luciferase transactivation. As described in U.S. patent
application Ser. No. 07/715,196, filed Jun. 14, 1991, entitled
Antisense Inhibition of RAS Oncogene and assigned commonly with
this application, the entire contents of which are herein
incorporated by reference, the ras oncogenes are members of a gene
family that encode related proteins that are localized to the inner
face of the plasma membrane. Ras proteins have been shown to be
highly conserved at the amino acid level, to bind GTP with high
affinity and specificity, and to possess GTPase activity. Although
the cellular function f ras gene products is unknown, their
biochemical properties, along with their significant sequence
homology with a class of signal-transducing proteins known as GTP
binding proteins, r G proteins, suggest that ras gene products play
a fundamental role in basic cellular regulatory functions relating
t the transduction of extracellular signals across plasma
membranes.
[0074] Three ras genes, designated H-ras, K-ras, and N-ras, have
been identified in the mammalian genome. Mammalian ras genes
acquire transformation-inducing properties by singl point mutations
within their coding sequences. Mutations in naturally occurring ras
oncogenes have been localized to cod ns 12, 13, and 61. The most
commonly detected activating ras mutation found in human tumors is
in codon 12 of the H-ras gen in which a base change from GGC to GTC
results in a glycine-to-valine substitution in the GTPase
regulatory domain of the ras protein product. This single amino
acid change is thought to abolish normal control f ras pr tein
function, thereby converting a normally regulat d cell pr tein to
ne that is continuously active. It is believed that such deregulati
n of normal ras protein function is responsible for the
transformation from normal to malignant growth.
[0075] The following examples and procedures illustrate th present
invention and are not intended to limit the same.
EXAMPLE 1
[0076] Oligonucleotide Synthesis:
[0077] Unsubstituted and substituted oligonucleotides were
synthesized on an automated DNA synthesizer (Applied Biosystems
model 380B) using standard phosphoramidate chemistry with oxidation
by iodine. For phosphorothioate oligonucleotides, the standard
oxidation bottle was replaced by 0.2 M solution of
3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for th step
wise thiation of the phosphite linkages. The thiation wait step was
increased to 68 sec and was followed by th capping step. After
cleavage from the CPG column and deblocking in concentrated
ammonium hydroxide at 55.degree. C. (18 hr), the oligonucleotides
were purified by precipitation twice out of 0.5 M NaCl solution
with 2.5 volumes ethanol. Analytical gel electrophoresis was
accomplished in 20% acrylamide, 8 M urea, 454 mM Tris-borate
buffer, pH=7.0. Oligonucleotides and phosphorothioates were judged
from polyacrylamide gel electrophoresis to be greater than 80%
full-length material.
EXAMPLE 2
[0078] Oligonucleotide Having .alpha. Oligonucleotide Regions
Flanking Central .beta. Oligonucleotide Region
[0079] A. .alpha.-.beta. Mixed Oligonucleotide Having
Non-Symmetrical 3'-3' and 5'-5' Likages
[0080] For the preparation of a 15 mer, a first region 4
nucleotides long of an .alpha. oligonucleotide is prepared as per
th method of Gagnor, et. al., Nucleic Acids Research 1987, 15,
10419 or n a DNA synthesizer utilizing the general protocols of
Exampl 1. Pr paration is from the 5' dir cti n towards the 3' dir
ction. The terminal 3' hydroxyl gr ups is d prot ct d. A normal
.beta. region f a DNA oligonucle tide 7 nucle tides long is added
in a 3' to 5' direction terminating in a free 5' hydroxyl group. A
further 4 nucleotide long region of .alpha. nucleotides is then
added in a 5' to 3' direction. The resulting 15 mer mixed
.alpha.-.beta.-.alpha. oligonucleotide includes a 3 atom 3'-3'
linkage between the first a region and the .beta. region and a 5
atom 5'-5' linkage between the second a region and th .beta.
region.
[0081] B. .alpha.-.beta. Mixed Oligonucleotide Having
Non-Symmetrical 3'-3' and 5'-5' Linkages
[0082] The procedure of Example 2-A is repeated except the
intermediate .beta. region is added as a phosphorothioate region by
substitution a thiation step for the normal oxidization step.
Thiation is conducted via use of the Beaucage Reagent, i. ., the
1,2-benzodithiole-3-one 1,1-dioxide of Example 1.
[0083] C. .alpha.-.beta. Mixed Oligonucleotide Having Symmetrical 4
Atom Linkages
[0084] For the preparation of a 17 mer, a first regi n 4
nucleotides long is of an .alpha.-oligonucleotide is prepared on
the DNA synthesizer as per the method of Gagnor, et. al., Nucl ic
Acids Research 1987, 15, 10419. Preparation is from the 5'
direction towards the 3' direction. The terminal 3' hydroxyl groups
is deprotected. A single nucleoside surrogate unit,
1.alpha.-thymidyl-3.beta.-hydroxymethyl-3.alpha.-methoxytrityloxyme-
thyl-cyclobutan amidite (prepared as per U.S. patent application
Ser. No. 808,201, identified above) is condensed on the terminal 3'
hydroxyl group of the a-oligonucleotide region in the normal manner
as per Example 1. The trityl hydroxyl gr up blocking group of the
cyclobutyl thymidine nucleoside surrogate is deblocked. A 7
nucleotide region of phosphorothioate 2'-deoxy .beta.-nucleotide
sequence is added on the synthesizer. Upon completion of the DNA
region of the macromolecule a
1.alpha.-thymidyl-2.beta.-hydroxy-3.alpha.-methoxytrityloxycyclobutane
unit activated as a normal phosphoramidite on the 2 hydroxy will b
c ndens d on the gr wing macromolecul in the same manner as is the
1.alpha.-thymidyl-3.beta.-hydroxymethyl-3.alpha.-methoxytrityloxym
thyl-cyclobutane moiety above. Following d blocking of the trityl
bl cking group of the nucleoside surrogate unit, a furth r 4
nucleotide stretch f .alpha.-oligonucleotides is added to complete
the macromolecule. Deblocking, removal from the support and
purification of the resulting macromolecule is conducted in th
normal manner.
EXAMPLE 3
[0085] Oligonucleotide Having 2'-Substituted Oligonucleotides
Regions Flanking Central 2'-Deoxy Phosphorothioate Oligonucleotide
Region
[0086] A 15 mer RNA target of the sequence 5'GCG TTT TTT TTT TGC G
3' was prepared in the normal manner on the DNA sequenc r using RNA
protocols. A series of phosphorothioate complementary
oligonucleotides having 2'-O-substituted nucleotides in regions
that flank 2'-deoxy region are prepar d utilizing 2'-O-substituted
nucleotide precursor prepared as per known literature preparations,
i.e., 2'-O-methyl, or as per the procedures of PCT application
PCT/US91/05720 or U.S. patent application Ser. Nos. 566,977 or
918,362. The 2'-O-substituted nucleotides are added as their
5'-O-dimethoxytrityl-3'-phosphoramidites in the normal manner on
the DNA synthesizer. The complementary oligonucleotides have the
sequence of 5' CGC AAA AAA AAA AAA ACG C 3'. The 2'-O-substituent
was located in CGC and CG regions of these oligonucleotides. The
following 2'-O-substituents are used: 2'-fluoro; 2'-O-methyl;
2'-O-propyl; 2'-O-allyl; 2'-O-aminopropoxy;
2'-O-(methoxyethoxyethyl), 2'-O-imidazolebutoxy and
2'-O-imidazolepropoxy. Additionally the same sequence is prepared
in both as a phosphodiester and a phosphorothioate. Following
synthesis the test compounds and the target compound are subjected
to a melt analysis to measure their Tm's and nucl as resistance as
per the protocols in the above referenced PCT application
PCT/US91/05720. The test sequences were found n t be substrates for
RNase H whereas as the corresponding target s quence is. Th se test
sequences will be nuclease stable and will have increase binding
affinity t the target compared to the phosph diester analogue.
EXAMPLE 4
[0087] Olig nucl tide Having 2'-5' Ph sph diester ligonucl tid
Regions Flanking a Central 2'-Deoxy 3'-5' Phosphorothi ate
Oligonucleotide Region
[0088] For the preparation of a 20 mer oligonucleotide, a first
region of 6 RNA nucleotides having 2'-5' linkages is prepared as
per the method of Kierzek, et. al., Nucleic Acids Research 1992,
20, 1685 on a DNA synthesizer utilizing the general protocols of
this reference. Upon completion of the 2'-5' linked region, a
2'-deoxy phosphorothioate region f 3'-5' linked DNA oligonucleotide
8 nucleotides long is added. A further 6 nucleotide long region of
2'-5' linkages is then added to complete the oligonucleotide having
mixed 2'-5' and 3'-5' linkages.
EXAMPLE 5
[0089] Macromolecule Having Regions of Cyclobutyl Surr gate
Nucleosides Linked By Phosphodiester Linkages Flanking a Central
2'-Deoxy 3'-5' Phosphorothioate Oligonucleotide Regi n
[0090] For the preparation of a 20 mer oligonucleotide, a first
region of 6 cyclobutyl surrogate nucleosides link d by
phosphodiester linkages is prepared as per Example 38 of U.S.
patent application Ser. No. 808,201 on a DNA synth sizer utilizing
the protocols of this reference. Upon completion f this region, a
2'-deoxy phosphorothioate region of a 3'-5' linked DNA
oligonucleotide 8 nucleotides long is added. A further region of 6
cyclobutyl surrogate nucleosides is then added to complete the
macromolecule.
EXAMPLE 6
[0091] Macromolecule Having Regions of Carbocyclic Surr gate
Nucleosides Linked By Phosphodiester Linkages Flanking a Central
2'-Deoxy Phosphorothioate Oligonucleotide Region
[0092] Carbocyclic nucleosid s are prepare as per the review
references cited in Borthwick, t al., T trah dron 1992, 48, 571.
The resulting carbocyclic nucleosides are blocked with a dimeth
xytrityl bl cking group in the normal manner. The c rresponding
phosphoramidites are prepared in th manner of Example 38 of U.S.
patent application Ser. No. 808,201 substituting the carbocyclic
nucleosides for the cyclobutyl nucleosides surrogates. For the
preparation of a 18 mer oligonucleotide, a first region of 4
carbocyclic nucleosides linked by phosphodiester linkages is
prepared on a DNA synthesizer utilizing the protocols of Example 1.
Up n completion of this region, a 2'-deoxy phosphorothioate 3'-5'
linked DNA oligonucleotide 8 nucleotides long is added. A further
region of 4 carbocyclic nucleotides is added to complete the
macromolecule.
EXAMPLE 7
[0093] Oligonucleotide Having 4'-Thionucleotide Regions Flanking a
Central 2'-Deoxy Phosphorothioate Oligonucleotide Region
[0094] In the manner of Example 6, a region of 4'-thionucleotides
is prepared as per the procedures of PCT patent application
PCT/US91/02732. Next a region of normal 2'-de xy phosphorothioate
nucleotides are added followed by a further region of the
4'-thionucleotides.
EXAMPLE 8
[0095] Macromolecule Having Peptide Nucleic Acids Regions Flanking
a Central 2'-Deoxy Phosphorothioate Oligonucleotide Region
[0096] A first region of peptide nucleic acids is prepar d as per
PCT patent application PCT/EP/01219. The peptide nucleic acids are
prepared from the C terminus towards the N terminus using monomers
having protected amine groups. Following completion of the first
peptide region, the terminal amine blocking group is removed and
the resulting amine reacted with a
3'-C-(formyl)-2',3'-dideoxy-5'-trityl nucleotide as prepared as per
the procedure of Vasseur, et. al., J. Am. Ch m. Soc. 1992, 114,
4006. The condensation of the amine with the aldehyde moi ty of the
C-formyl nucleoside is effected as per the conditions of the
Vasseur, ibid., t yield an intermediate oxime linkage. The oxime
linkag is r duced under reductive alkylation c nditions of Vass ur,
ibid., with HCHO/NaBH.sub.3CN/AcOH to yi ld th nucleoside c nnected
to the peptide nucleic acid via an methyl alkylated amine linkage.
An internal 2'-deoxy phosphorothioate nucleotide region is then
continued from this nucleoside as per the protocols of Example 1.
Peptide synthesis for the second peptide region is commenced by
reaction of the carboxyl and of the first peptide nucleic acid of
this second region with the 5' hydroxy of the last nucleotide of
the DNA region following removal of the dimethoxytrityl blocking
group on that nucleotide. Coupling is effected via DEA in pyridine
to form an ester linkage between the peptide and the nucleoside.
Peptide synthesis is then continued in the manner of patent
application PCT/EP/01219 t complete the second peptide nucleic acid
region.
EXAMPLE 9
[0097] Oligonucleotide Having 2'-Substituted Oligonucleotide Regi
ns Flanking a Central 2'-Deoxy Phosphoroselenate Oligonucleotide
Region
[0098] An oligonucleotide is prepared as per Example 3 utilizing
2'-O-methyl substituted nucleotides to prepare the flanking regions
and oxidization with 3H-1,2-benzothiaseleno-3-ol for introducing
the seleno moieties in the central regi n as per the procedure
reported by Stawinski, et al., T nth International Roundtable:
Nucleosides, Nucleotides and Th ir Biological Evaluation, Sep.
16-20, 1992, Abstracts f Papers, Abstract 80.
EXAMPLE 10
[0099] Oligonucleotide Having 2'-Substituted Oligonucleotide Regi
ns Flanking a Central 2'-Deoxy Phosphorodithioate Oligonucleotid
Region
[0100] An oligonucleotide is prepared as per Exampl 3 utilizing
2'-O-aminopropoxy substituted nucleotides to prepare the flanking r
gions and th procedures of Beaton, t. al., Chapter 5, Synthesis of
oligonucleotide pho phorodithioates, page 109, Oligonucl otid s and
Analogs, A Practical Approach, Eckstein, F., Ed.; The Practical
Appr ach Series, IRL Press, New York, 1991 to prepare the internal
phosphorodithioate region.
EXAMPLE 11
[0101] Oligonucleotide Having Boranophosphate Linked Oligonucle tid
Regions Flanking a Central 2'-Deoxy Phosphorothioate
Oligonucleotide Region
[0102] An oligonucleotide is prepared as per Exampl 3 utilizing the
procedures of published patent applicati n PCT/US/06949 to prepare
the flanking boranophosphate reqi ns and the procedures of Example
1 to prepare the central 2'-de xy phosphorothioate region.
EXAMPLE 12
[0103] Oligonucleotide Having 2'-Substituted Methyl Phosphonate
Linked Oligonucleotide Regions Flanking a Central 2'-Deoxy Phosph r
-thioate Oligonucleotide Region
[0104] 2-Fluoro nucleosides are prepared as per Example 3 and then
converted to nucleotides for the preparation of flanking
methylphosphonates linkages as per the procedures Miller et. al.,
Chapter 6, Synthesis of oligo-2'-deoxyribonucleoside
methylphosphonates, page 137, Oligonucleotides and Analogs, A
Practical Approach, Eckstein, F., Ed.; The Practical Appr ach
Series, IRL Press, New York, 1991. The central int rnal
phosphorothioate region is prepared as per Example 1 followed by
the addition of a further 2'-O-substituted methylphosph nate
region.
EXAMPLE 13
[0105] Oligonucleotide Having 2'-Substituted Methyl Phosphotriester
Linked Oligonucleotide Regions Flanking Central 2'-D xy
Phosphodiester Thymidine Oligonucleotide Region
[0106] 2-Fluoro nucleosides are prepared as per Example 3 and then
converted to nucleotid s for th pr paration of flanking r gions of
methyl phosphotriest r linkages as per the procedur s Miller, t.
al., Bioch mistry 1977, 16, 1988. A central internal phosphodiest r
regi n having 7 c nsecutiv thymidine nucl otid residues is prepar d
as per Example 1 followed by the addition of a further
2'-O-substituted methyl phosphotriester region.
EXAMPLE 14
[0107] Macromolecule Having Hydroxylamine Oligonucleoside Regi ns
Flanking a Central 2'-Deoxy Phosphorothioate Oligonucleotide
Region
[0108] A first flanking region of nucleosides alternately linked by
methylhydroxylamine linkages and phosphodiester linkages is
prepared as per the procedure of Vasseur, ibid. A central
2'-O-deoxy phosphorothioate oligonucleotide region is added as per
the procedure of Example 3 followed by a further flanking region
having the same linkages as the first region to complete the
macromolecule.
EXAMPLE 15
[0109] Macromolecule Having Hydrasine Linked Oligonucleoside Regi
ns Flanking a Central 2'-Deoxy Phosphorothioate Oligonucleotide
Region
[0110] A first flanking region of nucleosides linked by
methylhydrazine linkages is prepared as per the procedures of the
examples of patent application PCT/US92/04294. A central 2'-O-deoxy
phosphorothioate oligonucleotide region is add d as per the
procedure of Example 3 followed by a further flanking region having
the same linkages as the first region to compl t the
macromolecule.
EXAMPLE 16
[0111] Macromolecule Having Methysulfenyl Linked Oligonucleoside
Regions Flanking A Central 2'-Deoxy Phosphorothi ate
Oligonucleotide Region
[0112] A first flanking region of nucleosides linked by methylsulf
nyl linkages is prepared as per th procedur s f the xamples of
patent application PCT/US92/04294. A central 2'-O-deoxy ph
sphorothioat oligonucleotide region is add d as per the pr cedur of
Example 3 f llowed by a further flanking region having the same
linkages as the first region t complete the macromolecule.
EXAMPLE 17
[0113] Macromolecule Having Ethanediylimino Linked Oligonucleoside
Regions Flanking a Central 2'-Deoxy Phosphorothioate
Oligonucleotide Region
[0114] A first flanking region of nucleosides linked by
1,2-ethanediylimino linkages is prepared as per the procedures f
the examples of patent application PCT/US92/04294. A central
2'-O-deoxy phosphorothioate oligonucleotide region is added as per
the procedure of Example 3 followed by a further flanking region
having the same linkages as the first region to complete the
macromolecule.
EXAMPLE 18
[0115] Oligonucleotide Having Methylene Phosphonate Link d
Oligonucleotide Regions Flanking a Central 2'-D oxy
Phosphorothioate Oligonucleotide Region
[0116] A first flanking region of nucleosides linked by methylene
phosphonate linkages is prepared as per the procedur of the
examples of patent application PCT/US92/04294. A central 2'-O-deoxy
phosphorothioate oligonucleotide region is added as per the
procedure of Example 3 followed by a further flanking region having
the same linkages as the first region to complete the
macromolecule.
EXAMPLE 19
[0117] Macromolecule Having Nitrilomethylidyne Linked
Oligonucleoside Regions Flanking a Central 2'-Deoxy Phosphorothi
ate Oligonucleotide Region
[0118] A first flanking region of nucleosides linked by
nitrilomethylidyne linkages is prepared as per the procedures of th
exampl s of U.S. pat nt application Ser. No. 903,160. A central
2'-O-d oxy phosphorothioat oligonucleotid region is add d as per
the procedure of Example 3 followed by a further flanking region
having the same linkages as th first region to complete the
macromolecule.
EXAMPLE 20
[0119] Macromolecule Having Carbonate Linked Oligonucleoside Regi
ns Flanking a Central 2'-Deoxy Phosphorothioate Oligonucleotide
Region
[0120] A first flanking region of nucleosidts linked by carbonate
linkages is prepared as per the procedure of Mertes, et al., J.
Med. Chem. 1969, 12, 154. A central 2'-O-d oxy phosphorothioate
oligonucleotide region is added as per the procedure of Example 3
followed by a further flanking regi n having the same linkages as
the first region to complete th macromolecule.
EXAMPLE 21
[0121] Macromolecule Having Carbamate Linked Oligonucleoside
Regions Flanking a Central 2'-Deoxy Phosphorothioate
Oligonucleotide Region
[0122] A first flanking region of nucleosides linked by carbamate
linkages is prepared as per the procedure of Gait, et. al., J.
Chem. Soc. Perkin 1 1974, 1684. A central 2'-O-deoxy
phosphorothioate oligonucleotide region is added as per the
procedure of Example 3 followed by a further flanking region having
the same linkages as the first region to complete the
macromolecule.
EXAMPLE 22
[0123] Macromolecule Having Silyl Linked Oligonucleoside Regi ns
Flanking a Central 2'-Deoxy Phosphorothioate Oligonucleotide
Region
[0124] A first flanking region of nucleosides linked by silyl
linkages is prepared as per the procedure of Ogilvie, et al., Nucl
ic Acids R s. 1988, 16, 4583. A central 2'-O-deoxy phosphorothioate
oligonucleotide r gion is added as per the procedure of Example 3
foll wed by a further flanking r gion having the sam linkages as th
first region to c mplete the macromolecule.
EXAMPLE 23
[0125] Macromolecules Having Sulfide, Sulfoxide and Sulfone Linked
Oligonucleoside Regions Flanking a Central 2'-D oxy
Phosphorothioate Oligonucleotide Region
[0126] A first flanking region of nucleosides linked by sulfide,
sulfoxide and sulfone linkages is prepared as per the procedure of
Schneider, et al., Tetrahedron Letters 1990, 31, 335. A central
2'-O-deoxy phosphorothioate oligonucleotide region is added as per
the procedure of Example 3 followed by a further flanking region
having the same linkages as the first region to complete the
macromolecule.
EXAMPLE 24
[0127] Macromolecules Having Sulfonate Linked Oligonucleoside
Regions Flanking a Central 2'-Deoxy Phosphorothioate Oligonucleotid
Region
[0128] A first flanking region of nucleosides linked by sulfonate
linkages is prepared as per the procedure of Musicki, et al., J.
Org. Chem. 1991, 55, 4231. A central 2'-O-de xy phosphorothioate
oligonucleotide region is added as per the procedure of Example 3
followed by a further flanking region having the same linkages as
the first region to complete th macromolecule.
EXAMPLE 24
[0129] Macromolecules Having Sulfonamide Linked Oligonucleoside
Regions Flanking a Central 2'-Deoxy Phosphorothi ate
Oligonucleotide Region
[0130] A first flanking region of nucleosides linked by sulfonamide
linkages is prepared as per the procedure of Kirshenbaum, et. al.,
The 5th San Diego Conference: Nucl ic Acids: New Frontiers, Poster
abstract 28, Nov. 14-16, 1990. A central 2'-O-deoxy
phosphorothioate oligonucleotide r gion is added as per the pr
cedur of Example 3 followed by a further flanking region having the
same linkag s as the first region to complete th macr mol cule.
EXAMPLE 25
[0131] Macromolecules Having Formacetal Linked Oligonucleoside Regi
ns Flanking a Central 2'-Deoxy Phosphorothioate Oligonucle tide
Region
[0132] A first flanking region of nucleosides linked by formacetal
linkages is prepared as per the procedure of Matteucci, Tetrahedron
Letters 1990, 31, 2385,or Veeneman, t. al., Recueil des Trav. Chim.
1990, 109, 449. A central 2'-O-deoxy phosphorothioate
oligonucleotide region is added as per the procedure of Example 3
followed by a further flanking region having the same linkages as
the first region to complete the macromolecule.
EXAMPLE 26
[0133] Macromolecules Having Thioformacetal Linked Oligonucle side
Regions Flanking a Central 2'-Deoxy Phosphorothioate
Oligonucleotide Region
[0134] A first flanking region of nucleosides linked by
thioformacetal linkages is prepared as per the procedure of
Matteucci, et. al., J. Am. Chem. Soc. 1991, 113, 7767 or Matteucci,
Nucleosides & Nucleotides 1991, 10, 231. A central 2'-O-deoxy
phosphorothioate oligonucleotide region is added as per the
procedure of Example 3 followed by a further flanking region having
the same linkages as the first region to complete the
macromolecule.
EXAMPLE 27
[0135] Macromolecules Having Morpholine Linked Oligonucleoside Regi
ns Flanking a Central 2'-Deoxy Phosphorothioate Oligonucle tid
Region
[0136] A first flanking region of nucleosides linked by morpholine
linkages is prepared as per the procedure of U.S. Pat. No.
5,034,506. A central 2'-O-d xy phosphorothioate oligonucleotide
region is added as per th pr cedure of Example 3 followed by a
further flanking regi n having the same linkages as th first region
to c mplete the macromolecule.
EXAMPLE 28
[0137] Macromolecules Having Amide Linked Oligonucleoside Regi ns
Flanking a Central 2'-Deoxy Phosphorothioate Oligonucleotid
Region
[0138] A first flanking region of nucleosides linked by amide
linkages is prepared as per the procedure of U.S. patent
application Ser. No. 703,619 filed May 21, 1991 and related PCT
patent application PCT/US92/04305. A central 2'-O-deoxy
phosphorothioate oligonucleotide region is added as per the
procedure of Example 3 followed by a further flanking region having
the same linkages as the first region to complet the
macromolecule.
EXAMPLE 29
[0139] Macromolecules Having Ethylene Oxide Linked Oligonucle side
Regions Flanking a Central 2'-Deoxy Phosphodi ster Oligonucleotide
Region
[0140] A first flanking region of nucleosides linked by ethylene
oxide linkages is prepared as per the procedure of PCT patent
application PCT/US91/05713. A central. 2'-O-d oxy phosphodiester
oligonucleotide region three nucleotides long is added as per the
procedure of Example 1 followed by a further flanking region having
the same linkages as the first region to complete the
macromolecule.
EXAMPLE 30
[0141] Macromolecules Having 3,4-Dihydroxybutyl Linked Nucle base
Regions Flanking a Central 2'-Deoxy Phosphorothioat Oligonucleotide
Region
[0142] A first flanking region of nucleobases linked by
3,4-dihydroxybutyl linkages is prepared as per the procedure of
Augustyns, t. al., Nucl ic Acids R s arch 1991, 19, 2587. A central
2'-O-deoxy ph sphorothi ate olig nucl otide regi n is added as per
th procedure of Example 3 followed by a further flanking regi n
having the same linkages as the first region to complete the macr m
lecule.
EXAMPLE 31
[0143] Macromolecules Having Dihydroxyproproxymethyl Linked
Nucleobase Regions Flanking a Central 2'-Deoxy Phosphorothi ate
Oligonucleotide Region
[0144] A first flanking region of nucleobases linked by
dihydroxyproproxymethyl linkages is prepared as per th procedure of
Schneider, et al., J. Am. Chem. Soc. 1990, 112, 453. A central
2'-O-deoxy phosphorothioate oligonucleotide region 9 nucleotides
long is added as per the procedure of Example 3 followed by a
further flanking region having the same linkages as the first
region to complete the macromolecule.
[0145] Procedure 1
[0146] Ras-Luciferase Reporter Gene Assembly
[0147] The ras-luciferase reporter genes described in this study
were assembled using PCR technology. Oligonucle tide primers were
synthesized for use as primers for PCR cloning of the 5'-regions of
exon 1 of both the mutant (codon 12) and non-mutant (wild-type)
human H-ras genes. H-ras gene templates were purchased from the
American Type Culture Collection (ATCC numbers 41000 and 41001) in
Bethesda, Md. The oligonucleotide PCR primers
5'-ACA-TTA-TGC-TAG-CTT-TTT-GAG-TAA-ACT-TGT-GGG-GCA-GGA-GAC-CCT-GT-
-3' (sense), SEQ ID NO: 7, and
5'-GAG-ATC-TGA-AGC-TTC-TGG-ATG-GTC-AGC-GC-3- ' (antisense), SEQ ID
NO: 8, were used in standard PCR reactions using mutant and
non-mutant H-ras genes as templates. These primers are expected to
produce a DNA product of 145 base pairs corresponding to sequences
-53 to +65 (relative to the translational initiation site) of
normal and mutant H-ras, flanked by NheI and HindIII restriction
endonuclease sites. The PCR product was gel purified, precipitated,
washed and resuspended in water using standard procedures.
[0148] PCR primers f r the cl ning f the P. pyralis (firefly)
luciferase g ne were designed such that the PCR pr duct would code
for the full-l ngth luciferase pr tein with the exception of the
amino-terminal methionine residue, which would be replaced with two
amino acids, an amino-terminal lysine residue followed by a leucine
residue. Th oligonucleotide PCR primers used for the cloning of the
luciferase gene were
5'-GAG-ATC-TGA-AGC-TTG-AAG-ACG-CCA-AAA-ACA-TAA-AG-3' (sense), SEQ
ID NO: 9, and 5'-ACG-CAT-CTG-GCG-CGC-CGA-TAC-CGT-CGA-CCT-CCA-3'
(antisense), SEQ ID NO: 10, were used in standard PCR reactions
using a commercially available plasmid (pT3/T7-Luc) (Clontech),
containing the luciferase reporter gene, as a template. These
primers wer expected to yield a product of approximately 1.9 kb
corresponding to the luciferase gene, flanked by HindIII and BssHII
restriction endonuclease sites. This fragment was gel purified,
precipitated, washed and resuspended in water using standard
procedures.
[0149] To complete the assembly of the ras-luciferase fusion
reporter gene, the ras and luciferase PCR products were digested
with the appropriate restriction endonucleases and cloned by
three-part ligation into an expression vector containing the
steroid-inducible mouse mammary tumor virus promotor MMTV using the
restriction endonucleases NheI, HindIII and BssHII. The resulting
clone results in the insertion f H-ras 5' sequences (-53 to +65)
fused in frame with the firefly luciferase gene. The resulting
expression vector encodes a ras-luciferase fusion product which is
expressed under control of the steroid-inducible MMTV promoter.
[0150] Procedure 2
[0151] Transfection of Cells with Plasmid DNA:
[0152] Transfections were performed as described by Greenberg, M.
E. in Current Protocols in Molecular Biology, (Ausubel, et al.,
eds.), John Wiley and Sons, New York, with the following
modifications. HeLa cells were plated on 60 mm dishes at
5.times.10.sup.5 cells/dish. A total of 10 .mu.g of DNA was added t
each dish, of which 9 .mu.g was ras-luciferase r porter plasmid and
1 .mu.g was a vector expressing the rat glucocorticoid receptor
under contr l f the constitutive Rous sarcoma virus (RSV) promoter.
Calcium phosphate-DNA coprecipitates vere rem v d after 16-20 hours
by washing with Trim-buffered saline [50 Mm Tris-Cl (pH 7.5), 150
mM NaCl] containing 3 mM EGTA. Fr sh medium supplemented with 10%
fetal bovine serum was then add d to the cells. At this time, calls
ware pr*-treated with antisense oligonucleotides prior to
activation of reporter g ne expression by dexamethasone.
[0153] Procedure 3
[0154] Oligonucleotide Treatment of Cells:
[0155] Immediately following plasmid transfection, calls were
washed three times with Opti-MMD (Gibco), prewarmad to 37.degree.
C. Two ml of Opti-MEM containing 10 .mu.g/ml
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimet- hylammonium chloride
(DOTMA) (Bethesda Research Labs, Gaithersburg, Md.) was added to
ach dish and oligonucleotides were added directly and incubated for
4 hours at 37.degree. C. Opti-MEM was then removed and replaced
with the appropriate call growth medium containing oligonucle tid.
At this time, reporter gene expression was activated by treatment
of cells with dexamethasone to a final concentrati n of 0.2 .mu.M.
Cells were harvested 12-16 hours following steroid treatment.
[0156] Procedure 4
[0157] Luciferase Assays
[0158] Luciferase was extracted from cells by lysis with the
detergent Triton X-100, as described by Greenberg, M. E., in
current Protocols in Molecular Biology, (Ausubel, et al., eds.),
John Wiley and Sons, New York. A Dynatech ML1000 lumin meter was
used to measure peak luminescence upon additi n f luciferin (Sigma)
to 625 .mu.M. For each extract, lucif ras assays were performed
multiple times, using differing amounts of extract to ensur that
the data wer gath red in the linear range of the assay.
[0159] Procedure 5
[0160] Antisens Olig nucleotid Inhibiti n of ras-Luciferas Gene
Expression
[0161] A series of antisense phosphorothioate oligonucleotide
analogs targeted to the codon-12 point mutation of activated H-ras
were tested using the ras-luciferase reporter gene system described
in the foregoing examples. This series comprised a basic sequence
and analogs of that basic sequence. The basic sequence was of known
activity an reported in patent application Ser. No. 07/715,196
identified above. In both the basic sequence and its analogs, each
of the nucleotide subunits incorporated phosphorothioate linkages
to provide nuclease resistance. Each of the analogs incorporated
nucleotide subunits that contained 2'-O-methyl substituti ns and
2'-deoxy-erythro-pentofuranosyl sugars. In the analogs, a
sub-sequence of the 2'-deoxy-erythro-pentofuranosyl sugar
containing subunits were flanked on both ends by sub-sequences of
2'-O-methyl substituted subunits. The analogs differed from one
another with respect to the length of the sub-sequence f the
2'-deoxy-erythro-pentofuranosyl sugar containing nuclotides. The
length of these sub-sequences varied by 2 nucleotides between 1 and
9 total nucleotides. The 2'-deoxy-erythro-pentofuranosyl nucleotide
sub-sequences were centered at the point mutation of the codon-12
point mutation of the activated ras.
[0162] The base sequences, sequence reference numbers and sequence
ID numbers of these oligonucleotides (all are phosphorothioate
analogs) are shown in Table 1. In this table those nucleotides
identified with a .sup."M"contain a 2'-O-methyl substituent group
and the remainder of the nucleotides identified with .sub."D" are
2'-deoxy-erythro-pentofuranosyl nucleotides.
1TABLE 2 olig r f. no. ID NO: S quenc SEQ 2570
C.sub.dC.sub.dA.sub.d C.sub.dA.sub.dC.sub.d C.sub.dG.sub.dA.sub.d
C.sub.dG.sub.dG.sub.d C.sub.dG.sub.dC.sub.d C.sub.dC.sub.d 1 3975
C.sup.NC.sup.NA.sup.N C.sup.NA.sup.NC.sup.N C.sup.NG.sup.NA.sub.d
C.sup.NG.sup.NG.sup.N C.sup.NG.sup.NC.sup.N C.sup.NC.sup.N 2 3979
C.sup.NC.sup.NA.sup.N C.sup.NA.sup.NC.sup.N C.sup.NG.sub.dA.sub.d
C.sub.dG.sup.NG.sup.N C.sup.NG.sup.NC.sup.N C.sup.NC.sup.N 3 3980
C.sup.NC.sup.NA.sup.N C.sup.NA.sup.NC.sup.N C.sub.dG.sub.dA.sub.d
C.sub.dG.sub.dG.sup.N C.sup.NG.sup.NC.sup.N C.sup.NC.sup.N 4 3985
C.sup.NC.sup.NA.sup.N C.sup.NA.sup.NC.sub.d C.sub.dG.sub.dA.sub.d
C.sub.dG.sub.dG.sub.d C.sup.NG.sup.NC.sup.N C.sup.NC.sup.N 5 3984
C.sup.NC.sup.NA.sup.N C.sup.NA.sub.dC.sub.d C.sub.dG.sub.dA.sub.d
C.sub.dG.sub.dG.sub.d C.sub.dG.sup.NC.sup.N C.sup.NC.sup.N 6
[0163] FIG. 1 shows dose-response data in which cells were treated
with the phosphorothioate oligonucleotides of Tabl 1.
Oligonucleotide 2570 is targeted to the codon-12 point mutation of
mutant (activated) H-ras RNA. The other nucleotides have
2'-O-methyl substituents groups thereon to increase binding
affinity with sections of various lengths of inter-spaced
2'-deoxy-erythro-pentofuranosyl nucleotides. The control
oligonucleotide is a random phosphorothioate oligonucleotide
analog, 20 bases long. Results are expressed as percentage f
luciferase activity in transfected cells not treated with
oligonucleotide. As the figure shows, treatment of cells with
increasing concentrations of oligonucleotide 2570 resulted in a
dose-dependent inhibition of ras-luciferase activity in cells
expressing the mutant form of ras-luciferase. Oligonucleotide 2570
displays an approximate threefold selectivity toward the mutant
form of ras-luciferase as compared to the normal form.
[0164] As is further seen in FIG. 1, each of the olig-nucleotides
3980, 3985 and 3984 exhibited greater inhibition of ras-luciferase
activity than did oligonucleotide 2570. The greatest inhibition was
displayed by oligonucleotide 3985 that has a sub-sequence of
2'-deoxy-erythro-pentofur- anosyl nucleotides seven nucleotides
long. Oligonucleotide 3980, having a five nucleotide long
2'-deoxy-erythro-pentofuran syl nucl otide sub-sequence exhibited
the next gr atest inhibiti n follow d by oligonucleotide 3984 that
has a nine nucleotid 2'-deoxy-erythro-pentofura- nosyl nucleotide
sub-s quence.
[0165] FIG. 2 shows th results similar to FIG. 1 except it is in
bar graph form. Further seen on Figur 2 is the activity of
oligonucleotide 3975 and oligonucleotide 3979. These
oligonucleotides have sub-sequences of
2'-deoxy-erythro-pentofuranosyl nucleotides one and three
nucleotides l ng, respectively. As is evident from FIG. 2 neither
of the oligonucleotides having either the one nor the three 2'-de
xy-erythro-pentofuranosyl nucleotide sub-sequences sh wed
significant activity. There was measurable activity for the three
nucleotide sub-sequence oligonucleotide 3979 at the highest
concentration dose.
[0166] The increases in activity of oligonucleotides 3980, 3985 and
3984 compared to oligonucleotide 2570 is attributed to the increase
in binding affinity imparted to these compounds by the 2'-O-methyl
substituent groups located on the compounds and by the RNase H
activation imparted to these compounds by incorporation of a
sub-sequence of 2'-deoxy-erythro-pentofuranosyl nucleotides within
the main sequence of nucleotides. In contrast to the active
compounds of the invention, it is interesting to note that
sequences identical to those of the active oligonucleotides 2570,
3980, 3985 and 3984 but having phosphodiester linkages in stead of
the phosphorothioate linkages of the active oligonucleotides of the
invention showed no activity. This is attributed to these
phoaphodiester compounds being substrates for nucleases that
degrade such phosphodiester compounds thus preventing them
potentially activating RNase H.
Sequence CWU 1
1
10 1 17 DNA Artificial Sequence Oligonucleotide 1 ccacaccgac
ggcgccc 17 2 17 DNA Artificial Sequence Oligonucleotide 2
ccacaccgac ggcgccc 17 3 17 DNA Artificial Sequence Oligonucleotide
3 ccacaccgac ggcgccc 17 4 17 DNA Artificial Sequence
Oligonucleotide 4 ccacaccgac ggcgccc 17 5 17 DNA Artificial
Sequence Oligonucleotide 5 ccacaccgac ggcgccc 17 6 17 DNA
Artificial Sequence Oligonucleotide 6 ccacaccgac ggcgccc 17 7 47
DNA Artificial Sequence Oligonucleotide 7 acattatgct agctttttga
gtaaacttgt ggggcaggag accctgt 47 8 29 DNA Artificial Sequence
Oligonucleotide 8 gagatctgaa gcttctggat ggtcagcgc 29 9 35 DNA
Artificial Sequence Oligonucleotide 9 gagatctgaa gcttgaagac
gccaaaaaca taaag 35 10 33 DNA Artificial Sequence Oligonucleotide
10 acgcatctgg cgcgccgata ccgtcgacct cga 33
* * * * *