Non-enzymatic ligation of oligonucleotides

• US 5,476,930 A
• Filed: 01/23/1995
• Issued: 12/19/1995
• Est. Priority Date: 04/12/1993
• Status: Expired due to Term

• Current Assignee: Northwestern University
• Original Assignee: Northwestern University
• Inventors: Gryaznov, Sergei M., Letsinger, Robert L.
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): Kunz, Gary L.
• Application Number:
  US08/376,688
• Time in Patent Office: 330 Days
• Field of Search: 536/25.3, 536/24.33, 536/24.5
• US Class Current: 536/25.3
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2523/109 chemical ligation between nucleic acids

Abstract

A process for non-enzymatic convalent ligation of two oligonucleotides having the structure shown below: ##STR1## The spontaneous reaction of these above oligonucleotides produces a thiophosphorylacetylamino linkage as shown below: ##STR2## When carried out in the presence of a template, this reaction gives 90% yields within 20 minutes at micromolar concentrations.

Claims

1. Method of forming an oligonucleotide by:

a) reversibly binding at least two oligonucleotides at adjacent positions on an oligo- or polynucleotide including base units complementary to base units of the oligomers, wherein one of the oligonucleotides includes a nucleotide having a first reactive group with a 3'"'"' or 5'"'"' terminal bromoacetylamino proximate to a nucleotide of the other oligomer which having a second reactive group includes a 3'"'"' or 5'"'"' terminal phosphorothioate capable of spontaneously forming a covalent bond with the first reactive group; and

b) irreversibly covalently joining the oligonucleotides together through the first and second reactive groups having been brought in proximity to each other upon binding of the oligonucleotides on the polynucleotide in the absence of added reagent or enzyme to spontaneously form a thiophosphorylacetylamino bond through the reactive groups.

2. A method of forming an oligonucleotide of claim 1 wherein each of the oligomers consists of 8 to 20 nucleotides.

3. A method of forming an oligonucleotide of claim 1 wherein steps (a) and (b) occur in aqueous solution.

6. A method as set forth in claim 1 wherein the structure of the oligonucleotides is ##STR3##

7. A method as set forth in claim 1 wherein the structure of the oligonucleotides is ##STR4##

4. A method of forming an oligonucleotide by:

a) disposing at least two oligonucleotides in aqueous solution wherein one of the oligonucleotides has a 3'"'"' or 5'"'"' terminal bromoacetylamino group and the other of the nucleotides has a 3'"'"' or 5'"'"' terminal phosphorothioate group; and

b) covalently binding the oligonucleotides together through the α

-haloacyl group and the phosphothioate groups spontaneously forming a thiophosphorylacetylamino group therebetween.

5. A method of forming an oligonucleotide of claim 4 further including the step of (c) accelerating the reaction and carrying the reaction out to high completion by freezing the aqueous solution containing the oligonucleotides therein.