Methods of making nucleic acid molecules with reduced secondary structure

• US 20050032055A1
• Filed: 04/25/2003
• Published: 02/10/2005
• Est. Priority Date: 07/20/1999
• Status: Abandoned Application

• Current Assignee: Jeffrey R. Sampson
• Original Assignee: Jeffrey R. Sampson
• Inventors: Sampson, Jeffrey R.
• Application Number:
  US10/423,281
• Publication Number:
  US 20050032055A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/117 incorporating modified base

Abstract

The present invention provides a system for generating nucleic acid molecules having reduced levels of secondary structure compared to nucleic acid molecules of the same nucleotide sequence containing only naturally-occurring bases. Such molecules are referred to herein as “unstructured nucleic acids” (UNAs). UNAs have reduced levels of secondary structure because of their reduced ability to form intramolecular hydrogen bond base pairs between regions of substantially complementary sequence. Preferred UNAs, however, retain the ability to form intermolecular hydrogen bond base pairs with other nucleic acid molecules.

Claims

1. A method of synthesizing an unstructured nucleic acid with reduced secondary structure relative to a nucleic acid of substantially identical nucleotide sequence having naturally occurring bases, the method comprising steps of:

providing a template nucleic acid strand comprising at least two self-complementary sequence elements;

selecting a DNA polymerase for use in synthesizing a new unstructured nucleic acid strand complementary to the template nucleic acid strand so that the new unstructured strand contains at least two self-complementary sequence elements;

providing a collection of nucleotides that are substrates for the DNA polymerase, the collection being selected to allow for intermolecular base-pair interactions but not intermolecular base pair interactions; and

allowing extension by the DNA polymerase, so that the new unstructured nucleic acid strand is synthesized and the self-complementary sequence elements within the unstructured nucleic acid strand do not form intramolecular hybrids with one another but do form intermolecular hybrids with the template strand.

2. The method of claim 1 wherein the step of providing a collection comprises providing a collection of nucleotides comprising at least one purine analog and at least one pyrimidine analog that are not capable of forming a stably hydrogen bonded base pair with one another, but are each capable of forming a stable hydrogen bonded base pair with a nucleotide and a complementary position within the self-complementary sequence elements in the template strand.

3. The method of claim 1, wherein the step of providing a collection of nucleotides comprises providing a collection that includes nucleotides selected from the group consisting of:

2-aminodeoxyadenosine 5′

-triphosphate, 2-thiodeoxythymidine 5′

-triphosphate, deoxyinosine 5′

-triphosphate, deoxypyrrolopyrimidine 5′

-triphosphate, 2-thiodeoxycytidine 5′

-triphosphate, deoxyguanosine 5′

-triphosphate, deoxycytidine 5′

-triphosphate, deoxyadenosine 5′

-triphosphate, deoxythymidine 5′

-triphosphate, and combinations thereof.

4. The method of claim 1, wherein the new unstructured nucleic acid strand is at least 40 nucleotides in length.

5. The method of claim 1, wherein the new unstructured nucleic acid strand is at least 100 nucleotides in length.

6. The method of claim 1, wherein the new unstructured nucleic acid strand is at least 500 nucleotides in length.

7. The method of claim 1, further comprising a step of using the unstructured nucleic acid strand in an assay selected from the group consisting of:

a ligase assay, a polymerase extension assay, or a nucleic acid array hybridization assay.