Method of Determining Secondary Structure of a Nucleic Acid

Patent metadata

Field	Value
Publication number	US-2022098651-A1
Application number	US-202117482765-A
Country	US
Kind code	A1
Filing date	Sep 23, 2021
Priority date	Sep 30, 2020
Publication date	Mar 31, 2022
Grant date	—

Abstract

A method is disclosed for analyzing the secondary structure of a nucleic acid using a patch oligonucleotide and a probe oligonucleotide.

First claim

What is claimed is:

1 . A method of analyzing secondary structure of a nucleic acid comprising
combining the nucleic acid and a patch oligonucleotide and placing under hybridization conditions to hybridize the patch oligonucleotide to a patch binding site to form a patched nucleic acid; combining the patched nucleic acid with a probe oligonucleotide and placing under hybridization conditions to hybridize the probe oligonucleotide to a probe binding site to form a patched-probed nucleic acid; determining a first binding yield of the probe oligonucleotide to the patched nucleic acid; comparing the first binding yield to a second binding yield of the probe oligonucleotide to a naked nucleic acid; whereby the first binding yield being greater than or differing significantly from the second binding yield indicates that the patch binding site and the probe binding site are coupled and located on a common secondary structural element of the nucleic acid; and whereby the first binding yield and the second binding yield being similar indicates that the first binding site and the second binding site are independent and located on different secondary structural elements of the nucleic acid.

2 . The method of claim 1 wherein the nucleic acid is DNA or RNA.

3 . The method of claim 1 wherein the nucleic acid is a genome of a single-stranded RNA virus, a messenger RNA (mRNA), a long non-coding RNA (lncRNA), a ribosomal RNA (rRNA), a ribozyme, a transposable elements, a long terminal repeat (LTR) or a long interspersed nuclear element (LINE) retrotransposon.

4 . The method of claim 1 wherein binding to the patch binding site by the patch DNA oligonucleotide enhances binding of the probe oligonucleotide to the probe binding site.

5 . The method of claim 1 wherein the patch binding site and the probe binding site comprise complementary base pairings.

6 . The method of claim 1 wherein the nucleic acid is long RNA having 200 nucleotides in length or greater, between 200 nucleotides and 50,000 nucleotides in length, between 200 nucleotides and 30,000 nucleotides in length, between 200 nucleotides and 20,000 nucleotides in length, between 200 nucleotides and 10,000 nucleotides in length, between 500 nucleotides and 5,000 nucleotides in length, between 1000 nucleotides and 3,000 nucleotides in length, between 200 nucleotides and 5,000 nucleotides in length, between 200 nucleotides and 3,000 nucleotides in length, between 500 nucleotides and 10,000 nucleotides in length, or between 500 nucleotides and 3,000 nucleotides in length.

7 . The method of claim 1 wherein the first patch oligonucleotide and the second probe oligonucleotide are each at least 2 nucleotides in length, between 2 nucleotides and 200 nucleotides in length, between 2 nucleotides and 200 nucleotides in length, between 5 and 50 nucleotides in length, between 5 and 25 nucleotides in length, between 10 and 30 nucleotides in length, between 10 and 50 nucleotides in length, between 15 nucleotides and 25 nucleotides in length, between 12 and 48 nucleotides in length or between 12 and 24 nucleotides in length.

8 . The method of claim 1 wherein the nucleic acid and the patch oligonucleotide are combined in a reaction volume and the combination is under hybridization conditions to bind the patch oligonucleotide to a patch binding site of the nucleic acid.

9 . The method of claim 1 wherein the probe oligonucleotide is attached to a microarray and the patched nucleic acid is contacted to the microarray and the microarray is under hybridization conditions.

10 . The method of claim 1 wherein a plurality of probe oligonucleotides are attached to a microarray and the patched nucleic acid is contacted to the microarray and the microarray is under hybridization conditions.

11 . The method of claim 1 wherein a plurality of patch oligonucleotides are combined with the nucleic acid and the combination is under hybridization conditions to bind the plurality of patch oligonucleotides to respective patch binding sites of the nucleic acid to form a plurality-patched nucleic acid.

12 . The method of claim 1 wherein a plurality of patch oligonucleotides are combined with the nucleic acid and the combination is under hybridization conditions to bind the plurality of patch oligonucleotides to respective patch binding sites of the nucleic acid to form a plurality-patched nucleic acid;
thereafter the plurality-patched nucleic acid is contacted to a microarray having a plurality of probe oligonucleotides attached thereto and the microarray is hybridization conditions.

13 . The method of claim 11 wherein a binding yield is determined for each probe oligonucleotide.

14 . The method of claim 11 wherein a binding yield is determined for each probe oligonucleotide and compared to a binding yield for each probe oligonucleotide to the nucleic acid without the patch oligonucleotide to identify coupling between patch binding sites and probe binding sites.

16 . The method of claim 14 wherein the coupling between first binding sites and second binding sites indicates intramolecular base pairings between the patch binding sites and the probe binding sites.

16 . The method of claim 15 wherein the intramolecular base pairings are analyzed to generate secondary structure of the nucleic acid.

17 . The method of claim 1 wherein the nucleic acid is RNA.

18 . The method of claim 1 further comprising use of an RNA folding algorithm.

19 . The method of claim 1 with the proviso that an RNA folding algorithm is not used.

20 . The method of claim 1 where the nucleic acid and the patch oligonucleotide are combined using a microfluidic system.

21 . The method of claim 1 wherein the patched nucleic acid and the probe oligonucleotide are combined using a microfluidic system.

22 . The method of claim 1 wherein the patch oligonucleotide is DNA or RNA.

23 . The method of claim 1 wherein the probe oligonucleotide is DNA or RNA.

24 . The method of claim 1 wherein a plurality of patch oligonucleotides and a plurality of probe oligonucleotides are used to determine binding yields for a set of combinations of patch oligonucleotides and probe oligonucleotides.

25 . A combination comprising a nucleic acid, a patch oligonucleotide and a plurality of probe oligonucleotides.

26 . The combination of claim 25 wherein the plurality of probe oligonucleotides are attached to a microarray.

Assignees

• Harvard College

Inventors

• Garmann Rees F
• Chiang Timothy K
• Manoharan Vinothan N
• Kimchi Ofer
• Brenner Michael P

Classifications

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title