Closed nucleic acid structures

• US 10,184,147 B2
• Filed: 06/23/2016
• Issued: 01/22/2019
• Est. Priority Date: 12/19/2011
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Nelson, Norman C., Chelliserry, Jijumon
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/191,231
• Publication Number:
  US 20160298176A1
• Time in Patent Office: 943 Days
• 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 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid

Abstract

The invention provides compositions and methods for making closed nucleic acid structures in which one or both strands are continuous. The closed nucleic acid structures can be used as sequencing templates among other applications.

Claims

1. A method of forming a closed nucleic acid structure from target nucleic acid, comprising a method selected from the group consisting of:

(1) (a) denaturing a target nucleic acid;

(b) annealing a primer pair to opposing strands of the denatured target nucleic acid, each of the primers having a 5′

phosphate group, each of the primers having a 3′

segment and a 5′

segment, the 3′

segments of the primers being target-binding segments;

(c) extending the primers with a nucleic acid polymerase to obtain primer extended target nucleic acids, some of which have a strand with an overhanging 5′

segment from the first primer and others of which have an opposing strand with an overhanging 5′

segment from the second primer, wherein extension blocker oligonucleotides hybridizing to regions of the strands of the target nucleic acid 5′

to the regions hybridizing to the primers are used to align the 3′

ends of extended target nucleic acids with the 5′

nucleobase of the 3′

segments of the primers;

(d) denaturing the primer-extended target nucleic acids;

(e) annealing a first adaptor and a second adaptor, both having a 5′

region and a 3′

region, to the denatured primer-extended target nucleic acids to form an adaptor-capped nucleic acid having one strand comprising the 5′

segment from the first primer and an opposing strand comprising the 5′

segment from the second primer, and adaptors annealed to the 5′

segments of the primers at both ends of the adaptor-capped nucleic acid;

wherein the 3′

region of the adaptor comprises a stem-loop structure, the 5′

region of the adaptor having a 5′

phosphate group, and the 5′

region of the first adaptor is complementary to the 5′

segment of the first primer and the 5′

region of the second adaptor is complementary to the 5′

segment of the second primer; and

(f) contacting the adaptor-capped nucleic acid with a ligase which seals a nick in the adaptor-capped nucleic acid, thereby forming a closed nucleic acid structure;

(2) (a) denaturing a target nucleic acid;

(b) annealing a primer pair to opposing strands of the denatured target nucleic acid, each of the primers having a 3′

segment and a 5′

segment, the 3′

segments of the primers being target-binding segments, the 5′

segments being complementary in opposing orientations, a first of the primers having a 5′

phosphate group and the second of the primers lacking a 5′

phosphate and/or having a shorter 5′

segment than the first primer;

(c) extending the primers with a nucleic acid polymerase to obtain primer-extended target nucleic acids, some of which have a strand with an overhanging 5′

segment from the first primer and others of which have an opposing strand with an overhanging 5′

segment from the second primer;

(d) denaturing the primer-extended target nucleic acids;

(e) annealing strands of the denatured primer-extended target nucleic acids having overhanging 5′

segments, wherein strands and opposing strands anneal to one another and circularize by annealing of the 5′

segments thereby forming a closed nucleic acid structure; and

(f) contacting the closed nucleic acid structure with a ligase which seals a nick between the 5′

phosphate group of the first primer and an adjacent 3′

hydroxyl group leaving the closed nucleic acid structure with a single nick or gap between the 5′

segment of the second primer and a 3′

hydroxyl of an adjacent nucleotide;

(3) (a) contacting a target nucleic acid with a primer pair under amplification conditions, each of the primers having a 5′

phosphate group, each of the primers having a 3′

segment and a 5′

segment, the 3′

segments of the primers being target-binding segments;

the first 3′

to 5′

nucleobase unit in the 5′

segment is an extension blocker;

wherein the extension blocker blocks nucleobase unit extension by a nucleic acid polymerase when the 5′

segment serves as a template for nucleobase unit extension;

thereby forming an amplified target nucleic acid having a strand comprising an overhanging 5′

segment from the first primer and an opposing strand comprising an overhanging 5′

segment from the second primer;

(b) annealing a first adaptor and a second adaptor, both having a 5′

region and a 3′

region, to the amplified target nucleic acids to form an adaptor-capped nucleic acid, wherein the 3′

region of the adaptor comprises a stem-loop structure, the 5′

region of the adaptor having a 5′

phosphate group, and the 5′

region of the first adaptor is complementary to the 5′

segment of the first primer and the 5′

region of the second adaptor is complementary to the 5′

segment of the second primer; and

(c) contacting the adaptor-capped nucleic acid with a ligase which seals a nick in the adaptor-capped nucleic acid thereby forming a closed nucleic acid structure; and

(4) (a) contacting a target nucleic acid with a primer pair under amplification conditions, each of the primers having a 3′

segment and a 5′

segment, the 3′

segments of the primers being target-binding segments, the 5′

segments being complementary in opposing orientations, a first of the primers having a 5′

phosphate group and the second of the primers lacking a 5′

phosphate group and/or having a shorter 5′

segment than the first primer;

the first 3′

to 5′

nucleobase unit in the 5′

segment is an extension blocker;

wherein the extension blocker blocks nucleobase unit extension by a nucleic acid polymerase when the 5′

segment serves as a template for nucleobase unit extension, thereby forming an amplified target nucleic acids having a strand comprising an overhanging 5′

segment from the first primer and an opposing strand comprising an overhanging 5′

segment from the second primer (b) annealing an overhanging 5′

segment from the first primer of one strand of the amplified target nucleic acid and an overhanging 5′

segment from the second primer of the opposing strand of the same amplified target nucleic acid thereby forming an annealed target nucleic acid; and

(c) contacting the annealed target nucleic acid with a ligase which seals a nick between the 5′

phosphate group of the first primer and an adjacent 3′

hydroxyl group leaving the closed nucleic acid structure with a single nick or gap between the 5′

segment of the second primer and a 3′

hydroxyl of an adjacent nucleotide.

2. The method of claim 1, wherein the extension blocker at (3) or at (4) is a modified nucleobase unit that blocks nucleobase unit extension by a nucleic acid polymerase when the 5′

segment serves as a template for nucleobase unit extension in the absence of a partnering nucleobase unit that base pairs with the extension blocker, and the nucleobase unit extension is not blocked by the extension blocker in the presence of a partnering nucleobase unit that base pairs with the extension blocker; and

wherein the amplification conditions do not contain a partnering nucleobase unit.

3. The method of claim 1, wherein the extension blocker at (3) or at (4) is a reversibly modified nucleobase that blocks nucleobase unit extension by a nucleic acid polymerase when the 5′

segment serves as a template for nucleobase unit extension;

or, wherein the extension blocker at (3) or at (4) is one or more ribonucleotide units and the nucleic acid polymerase is a DNA dependent DNA polymerase.

4. The method of claim 1, wherein the extension blocker at (3) or at (4) is isoC and the partnering nucleobase unit is isoG;

or wherein the extension blocker at (3) or at (4) is isoG and the partnering nucleobase unit is isoC.

5. The method of claim 1, wherein the extension blocker at (3) or at (4) is selected from the group consisting of:

nucleotides with hydrolysis resistant modifications at the 2′

carbon atom, nucleotides with 0-linked triisopropylsilyl groups at the 2′

carbon atom, nucleotides with 0-linked tertButyl-dimethylsilyl groups al the 2′

carbon atom;

nucleotides with 0-linked alkyl groups at the 2′

carbon atom, and an A, C, T/U, or G nucleotide with a —

OSiC6 group at the 2′

carbon atom.

6. The method of claim 5, wherein the blocking group member of the extension blocker nucleotide is removed so that nucleobase unit extension is not blocked by the extension blocker.

7. The method of claim 1, wherein at (3) or at (4) an RNA dependent DNA polymerase is used so that nucleobase unit extension is not blocked by the extension blocker.

8. The method of claim 1, wherein the closed nucleic acid at (3) or at (4) is used in a reaction that includes the step of performing a nucleobase unit extension by a nucleic acid polymerase, and wherein the extension blocker does not block nucleobase unit extension by a nucleic acid polymerase.

9. The method of claim 1, wherein the closed nucleic acid structure is used in a sequencing reaction.

10. The method of claim 9, wherein the closed nucleic acid structure at (3) or at (4) is used in a sequencing reaction and wherein nucleobase unit extension blocking by the extension blocker is reversed.