Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases

• US 5,432,272 A
• Filed: 10/09/1990
• Issued: 07/11/1995
• Est. Priority Date: 10/09/1990
• Status: Expired due to Term

• Current Assignee: Steven A. Benner
• Original Assignee: Steven A. Benner
• Inventors: Benner, Steven A.
• Primary Examiner(s): Walsh, Stephen G.
• Assistant Examiner(s): WANG, GIAN
• Application Number:
  US07/594,290
• Time in Patent Office: 1,736 Days
• Field of Search: 536/25, 536/23, 536/24, 536/26, 536/27, 536/29, 536/28, 536/25.3, 536/25.33, 435/91, 435/91.1, 435/91.51, 435/91.41
• US Class Current: 536/25.3
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The disclosure describes a method for incorporating into double stranded DNA and RNA base pairs composed of pairing units that fit the Watson-Crick geometry in that they involve a monocyclic six membered ring pairing with a fused bicyclic heterocyclic ring system composed of a five member ring fused with a six membered ring, with the orientation of the heterocycles with respect to each other and with respect to the backbone chain analogous to that found in DNA and RNA, but with a pattern of hydrogen bonds holding the base pair together different from that found in the AT and GC base pairs (a "non-standard base pair").

Claims

1. A method for incorporating into a DNA or RNA oligonucleotide chain at least one nucleotide unit bearing a heterocyclic base selected from the group consisting of the structural formulae of FIG. 4, wherein --R designates the point of attachment of the base to position 1 of a ribose or deoxyribose ring, X is either a nitrogen atom or a carbon atom bearing a substituent Z, Z is either a hydrogen, an unfunctionalized lower alkyl chain, or a lower alkyl chain bearing an amino, carboxyl, hydroxyl, thiol, aryl, indole, or imidazolyl group, Y is either N or CH, and each ring contains no more than three nitrogens consecutively bonded, that consists of synthesizing a template that is an oligonucleotide containing one or more nucleotide subunits bearing a heterocyclic base selected from the group consisting of the structural formulae of FIG. 4, dissolving this template in buffered aqueous solution, adding to the solution nucleoside triphosphates complementary to the nucleotide subunits in the template, adding to this solution a solution of a DNA or RNA polymerase, and incubating said mixture of the solutions such that an oligonucleotide chain complementary to the template and incorporating said at least one oligonucleotide unit bearing a heterocyclic base selected from the group consisting of the structural formulae of FIG. 4 is synthesized.

2. The method of claim 1 wherein said heterocyclic base is selected from the group consisting of 2,6-diaminopyrimidine, xanthine, 2,6-diaminopyrazine, 1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione, iso-guanine, and iso-cytosine.

3. The method of claim 1 wherein said polymerase is selected from the group consisting of the Klenow fragment of DNA polymerase I, T7 RNA polymerase, and AMV reverse transcriptase.

4. The method of claim 1 wherein said heterocyclic base is iso-cytosine incorporated opposite iso-guanine in a template.

5. The method of claim 1 wherein said heterocyclic base is iso-guanine incorporated opposite iso-cytosine in a template.

6. The method of claim 1 wherein said heterocyclic base is 2,6-diaminopyrimidine or 2,6-diaminopyrazine incorporated opposite 1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione or xanthine in a template.

7. The method of claim 1 wherein said heterocyclic base is 1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione or xanthine incorporated opposite either 2,6-diaminopyrimidine or 2,6-diaminopyrazine in a template.