US5849482A
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Crosslinking oligonucleotides
Assignee: Epoch Biosciences Inc
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Filing: 1995-06-07
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Granted: 1998-12-15
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Expires: 2015-06-07
Abstract
Abstract Oligonucleotides (ODNs) include a sequence that is complementary to a target sequence in single stranded RNA, or single or double stranded DNA, and an alkylating function which after hybridization alkylates the target sequence. ODNs adapted for alkylating single stranded RNA, such as messenger RNA, are complementary to the target sequence in the Watson Crick sense. ODNs adapted for alkylating double stranded DNA have at least two alkylating functions and are complementary to the target sequence in the Hoogsteen or reverse Hoogsteen sense. With these ODNs both strands of the target sequence are alkylated. A third class of ODNs have at least approximately 26 nucleotide units in a continous sequence which are complementary to the target sequence of double stranded DNA, and the alkylating function is covalently attached to a nucleotide unit in the continuous sequence. Alkylation or cross-linking with this class of ODNs occurs in the presence of a recombinase enzyme.
Inventors
- 👤 Rich B. Meyer, Jr.
- 👤 Howard B. Gamper
- 👤 Igor V. Kutyavin
- 👤 Alexander A. Gall
- 👤 Charles R. Petrie
- 👤 John C. Tabone
- 👤 Gerald D. Hurst
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 22:46
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/113; A; A61; A61P; A61P31/00; A61P35/00; C07; C07H; C07H21/00; C12N15/10; C12N15/102; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6839; A61K; A61K38/00; C12N2310/00; C12N2310/10; C12N2310/15; C12N2310/153; C12N2310/30; C12N2310/31; C12N2310/311; C12N2310/33; C12N2310/335; C12N2310/35; C12N2310/351; C12N2310/3511
Claims Analysis
Total Claims: 7
Claim 1
Independent Claim
255 words
A process for cross-linking with a target sequence in double stranded DNA, the process comprising: contacting in the presence of a recombinase enzyme the double stranded DNA having the target sequence with an oligonucleotide of the formula ##STR10## where B--R--P* represents a nucleotide building block of the oligonucleotide which is substituted or unsubstituted with a reporter group and lacks or includes a radioactive label; B in said B--R--P* represents a heterocyclic base component of the nucleotide, R in said B--R--P* is selected from 2-deoxyribofuranosyl, 2-O-alkylribofuranosyl, and 2-deoxy-2-fluororibofuranosyl, and P* in said B--R--P* represents a phosphate group including a phosphate monoester, phosphate diester or phospate triester group, or P* represents a monothioate or dithioate analog of said phosphate groups; (A--(L) v ) w represents an electrophilic alkylating group wherein L is a leaving group, A is a group that covalently links L to the oligonucleotide and A--L is substantially inert in a cross-linking reaction under conditions that permit hybridization with the target sequence of double stranded DNA, and A--L reacts substantially only after hybridization with the target sequence has occurred; n is an integer with the values between 26 and approximately 300, v is 1 or 2; w is between 1-10, and the oligonucleotide includes a continuous sequence of at least approximately 26 nucleotide units which sequence is complementary in the Watson Crick sense to the a target sequence in the double stranded DNA, and wherein at least one A--(L) n group is covalently attached to the continuous sequence that is complementary to the target sequence.
Claim 2
Dependent Claim
255 words
A process for cross-linking with a target sequence in double stranded DNA, the process comprising: contacting in the presence of a recombinase enzyme the double stranded DNA having the target sequence with an oligonucleotide of the formula ##STR10## where B--R--P* represents a nucleotide building block of the oligonucleotide which is substituted or unsubstituted with a reporter group and lacks or includes a radioactive label; B in said B--R--P* represents a heterocyclic base component of the nucleotide, R in said B--R--P* is selected from 2-deoxyribofuranosyl, 2-O-alkylribofuranosyl, and 2-deoxy-2-fluororibofuranosyl, and P* in said B--R--P* represents a phosphate group including a phosphate monoester, phosphate diester or phospate triester group, or P* represents a monothioate or dithioate analog of said phosphate groups; (A--(L) v ) w represents an electrophilic alkylating group wherein L is a leaving group, A is a group that covalently links L to the oligonucleotide and A--L is substantially inert in a cross-linking reaction under conditions that permit hybridization with the target sequence of double stranded DNA, and A--L reacts substantially only after hybridization with the target sequence has occurred; n is an integer with the values between 26 and approximately 300, v is 1 or 2; w is between 1-10, and the oligonucleotide includes a continuous sequence of at least approximately 26 nucleotide units which sequence is complementary in the Watson Crick sense to the a target sequence in the double stranded DNA, and wherein at least one A--(L) n group is covalently attached to the continuous sequence that is complementary to the target sequence.
Claim 3
Dependent Claim
10 words
The process of claim 1 which is performed in vitro.
Claim 4
Dependent Claim
16 words
The process of claim 1 wherein the oligonucleotide has no more than approximately 60 nucleotide units.
Claim 5
Dependent Claim
171 words
The process of claim 1 wherein the A--(L) v is a group selected from the groups consisting of --(CH 2 ) q --Y--(CH 2 ) m --L, (CH 2 ) q --CO--CH 2 --L, --(CH 2 ) q --NH--CO--(CH 2 ) m --(X) n' --N(R 1 )--(CH 2 ) p --L, and --(CH 2 ) q' --O--(CH 2 ) q" --NH--CO--(CH 2 ) m --(X) n , --N(R 1 )--(CH 2 ) p --L where each of m and q is independently 0 to 8, inclusive, q' is 3 to 7 inclusive, q" is 1 to 7 inclusive, Y is a linking group derived from a bifunctional molecule having a hydrocarbyl backbone and having at each end a functionality selected from --NH 2 , --OH, SH, --COOH and C.tbd.CH, and X is phenyl, or phenyl substituted with chloro, bromo, lower alkyl or lower alkoxy groups, n' is 0 or 1, p is an integer from 1 to 6, and R 1 is H, lower alkyl or (CH 2 ) p --L.
Claim 6
Dependent Claim
78 words
The process of claim 1 wherein the A--(L) v group is selected from the groups consisting of --(CH 2 ) 3 O(CH 2 ) 2 NHCO(CH 2 ) 3 --C 6 H 4 --N--(CH 2 CH 2 Cl) 2 --(CH 2 ) 3 NHCO(CH 2 ) 3 --C 6 H 4 --N--(CH 2 CH 2 Cl) 2 , and --(CH 2 ) 6 NHCO(CH 2 ) 3 --C 6 H 4 --N--(CH 2 CH 2 Cl) 2 .
Claim 7
Dependent Claim
35 words
The process of claim 4 wherein the A--(L) v group is covalently attached to a nucleotide unit which is internal within the continuous sequence that is complementary to the target sequence of double stranded DNA.
References Cited (Backward Citations)
No citation records indexed.
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