Crosslinking oligonucleotides

• US 5,849,482 A
• Filed: 06/07/1995
• Issued: 12/15/1998
• Est. Priority Date: 09/28/1988
• Status: Expired due to Fees

• Current Assignee: Microprobe Corporation
• Original Assignee: Epoch Pharmaceuticals, Inc.
• Inventors: Hurst, Gerald D., Gall, Alexander A., Tabone, John C., Meyer, Rich B. Jr., Petrie, Charles R., Kutyavin, Igor V., Gamper, Howard B.
• Primary Examiner(s): LeGuyader, John L.
• Application Number:
  US08/485,611
• Time in Patent Office: 1,287 Days
• Field of Search: 514/44, 536/24.3, 536/24.5, 536/24.31, 536/24.32, 536/18.4, 536/124, 536/23.1, 435/6, 435/172.1, 435/172.3, 435/375, 436/94, 436/183
• US Class Current: 435/6.12
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/153 with the aid of a protein, e.g. recombinase
• CPC Class Codes: C12N 2310/311 Phosphotriesters
• CPC Class Codes: C12N 2310/335 Modified T or U
• CPC Class Codes: C12N 2310/3511 intercalating or cleaving agent
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine

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.

Claims

1. 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.

2. The process of claim 1 which is performed in vitro.

3. The process of claim 1 wherein the oligonucleotide has no more than approximately 60 nucleotide units.

4. The process of claim 1 wherein the A--(L)_v is a group selected from the groups consisting of

--(CH₂)_q --Y--(CH₂)_m --L, (CH₂)_q --CO--CH₂ --L,

--(CH₂)_q --NH--CO--(CH₂)_m --(X)_n'"'"' --N(R₁)--(CH₂)_p --L, and

--(CH₂)_q'"'"' --O--(CH₂)_q" --NH--CO--(CH₂)_m --(X)_n, --N(R₁)--(CH₂)_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 --NH₂, --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 R₁ is H, lower alkyl or (CH₂)_p --L.

5. The process of claim 1 wherein the A--(L)_v group is selected from the groups consisting of

--(CH₂)₃ O(CH₂)₂ NHCO(CH₂)₃ --C₆ H₄ --N--(CH₂ CH₂ Cl)₂

--(CH₂)₃ NHCO(CH₂)₃ --C₆ H₄ --N--(CH₂ CH₂ Cl)₂, and

--(CH₂)₆ NHCO(CH₂)₃ --C₆ H₄ --N--(CH₂ CH₂ Cl)₂.

6. 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.