Closed nucleic acid structures
View Patent ↗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.
1. 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.
2. The method of claim 1 , 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.
3. The method of claim 1 , wherein the 3′ blocked oligonucleotide comprises an affinity label.
4. The method of claim 3 , wherein the affinity label is biotin.
5. The method of claim 1 , wherein the 3′ blocked oligonucleotide is linked to a solid support.
6. The method of claim 1 , wherein the 3′ blocked oligonucleotide is separated from the 3′ extended nucleic acid strand by denaturation and purification.