CLOSED NUCLEIC ACID STRUCTURES

• US 20130203123A1
• Filed: 12/19/2012
• Published: 08/08/2013
• 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
• Granted Patent: US 9,404,147 B2
• Time in Patent Office: Days
• US Class Current: 435/91.52
• 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

1-129. -129. (canceled)

130. A method of forming a closed nucleic acid structure comprising a target nucleic acid, the method comprising:

(a) providing a denatured target nucleic acid, each strand of the denatured target nucleic acid having a 5′

phosphate and a 3′

segment;

(b) contacting the strands of the denatured target nucleic acid with a pair of 3′

blocked oligonucleotides, each oligonucleotide having a 3′

segment and a 5′

segment, the 3′

segments being complementary to the 3′

segments of the strands of the denatured target nucleic acid;

(c) annealing the 3′

segments of the 3′

blocked oligonucleotides to the 3′

segments of the denatured target nucleic acid strands, whereby each 3′

blocked oligonucleotide provides a template for extension of the denatured target nucleic acid strand to which it is annealed;

(d) extending the denatured target nucleic acid strands to form 3′

extended nucleic acid strands, each 3′

extended nucleic acid strand having from 5′

-3′

a 5′

phosphate group, a strand of the target nucleic acid, a 3′

extension complementary to the 5′

segment of a 3′

blocked oligonucleotide, and a 3′

hydroxyl group;

(e) contacting a duplex nucleic acid intermediate formed by annealing of the 3′

extended nucleic acid strands to one another with a pair of stem-loop adaptors, each adaptor having a 3′

segment and a 5′

segment, the 5′

segments each having a 5′

phosphate group and a sequence capable of forming a stem-loop structure and the 3′

segments being complementary to the 3′

extensions of the duplex nucleic acid intermediate and ending in a 3′

hydroxyl group;

(f) annealing the 3′

segments of the stem-loop adaptors to the 3′

extensions of the duplex target nucleic acid; and

(g) providing a ligase that seals nicks between the ends of the stem-loop adaptors and the ends of duplex target nucleic acid, thereby forming a closed nucleic acid structure including the two strands of the target nucleic acid joined to one another by stem-loop structures.

131. The method of claim 130, wherein following step (d), the method comprises:

(i) separating the 3′

blocked oligonucleotides from the 3′

extended nucleic acid strands; and

(ii) allowing intermolecular hybridization between the 3′

extended nucleic acid strands;

thereby forming a duplex nucleic acid intermediate having at each end a 5′

phosphate group and a single-stranded 3′

extension ending in a 3′

hydroxyl group, wherein the 3′

extensions are complementary to the 5′

segments of the 3′

blocked oligonucleotides.

132. The method of claim 130, wherein the 3′

blocked oligonucleotides contain deoxyribo-uracil and are separated from the extended target nucleic acid strands by digestion with a uracil DNA glycosidase (UNG) and an endonuclease.

133. The method of claim 130, wherein the 3′

blocked oligonucleotides comprise an affinity label.

134. The method of claim 133, wherein the affinity label is biotin.

135. The method of claim 130, wherein the 3′

blocked oligonucleotides are linked to a solid support.

136. The method of claim 130, wherein the 3′

blocked oligonucleotides are separated from the extended nucleic acid strands by denaturation and purification.

137. The method of claim 130, wherein the 5′

and 3′

segments of the stem-loop adaptors are linked by a segment consisting of 0 to 4 thymine nucleobase units.

138. A method of forming a closed nucleic acid structure comprising a target nucleic acid, the method comprising:

(a) providing a denatured target nucleic acid, at least one strand of the denatured target nucleic acid having a 5′

phosphate, a 5′

segment, and a 3′

segment;

(b) contacting the strand of the denatured target nucleic acid with a 3′

blocked oligonucleotide having a 5′

segment, a 3′

segment, and an intervening segment that links the 5′ and

3′

segments, the 3′

segment being complementary to the 3′

end segment of the denatured target nucleic acid strand and the intervening segment having a common sequence with the 5′

segment of the denatured target nucleic acid strand;

(c) annealing the 3′

segment of the 3′

blocked oligonucleotide to the 3′

segment of the denatured target nucleic acid strand, whereby the 3′

blocked oligonucleotide provides a template for extension of the denatured target nucleic acid strand;

(d) extending the denatured target nucleic acid strand to form a 3′

extended nucleic acid strand having from 5′

-3′

a 5′

phosphate group, the target nucleic acid strand, a 3′

extension complementary to the 5′ and

intervening segments of the 3′

blocked oligonucleotide, and a 3′

hydroxyl group;

(e) separating the 3′

blocked oligonucleotide from the 3′

extended nucleic acid strand;

(f) contacting the 3′

extended nucleic acid with a stem-loop adaptor having a 3′

segment and a 5′

segment, the 5′

segment having a 5′

phosphate group and a sequence capable of forming a stem-loop structure and the 3′

segment having a common sequence with the 3′

segment of the 3′

blocked oligonucleotide and ending in a 3′

hydroxyl group;

(g) allowing the 3′

extended nucleic acid strand to intermolecularly hybridize with the stem-loop adaptor and intramolecularly hybridize with itself;

thereby forming a nicked intermediate wherein the 3′

extended nucleic acid strand has a stem-loop structure, the 5′

phosphate group of the 3′

extended nucleic acid strand is separated from the 3′

hydroxyl group of the stem-loop adaptor by a nick, and the 5′

phosphate group of the stem-loop adaptor is separated from the 3′

hydroxyl group of the 3′

extended nucleic acid strand by a nick; and

(h) providing a ligase that seals nicks between the ends of the stem-loop adaptor and the ends of the 3′

extended nucleic acid, thereby forming a closed nucleic acid structure including the strand of the target nucleic acid circularized by a stem-loop structure.

139. The method of claim 138, wherein the 3′

blocked oligonucleotide contains deoxyribo-uracil and is separated from the extended target nucleic acid strand by digestion with a uracil DNA glycosidase (UNG) and an endonuclease.

140. The method of claim 138, wherein the 3′

blocked oligonucleotide comprises an affinity label.

141. The method of claim 140, wherein the affinity label is biotin.

142. The method of claim 138, wherein the 3′

blocked oligonucleotide is linked to a solid support.

143. The method of claim 138, wherein the 3′

blocked oligonucleotide is separated from the 3′

extended nucleic acid strand by denaturation and purification.

144-162. -162. (canceled)