Closed nucleic acid structures
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 segment of a target nucleic acid, the method comprising:
(a) contacting the target nucleic acid with a primer pair under amplification conditions, each of the primers of the pair including a 5′ phosphate group and a 3′ segment, the 3′ segments of the primers being target-binding segments; thereby forming an amplified nucleic acid comprising duplex target nucleic acid flanked by the primers of the pair duplexed with their complementary segments, wherein each strand of the amplified nucleic acid includes a 5′ phosphate group and a 3′ hydroxyl group;
(b) denaturing the amplified nucleic acid and contacting a strand of the denatured amplified nucleic acid with a stem-loop adaptor having a 5′ phosphate group, a 5′ segment, a 3′ segment having a stem-loop structure, and a 3′ hydroxyl group, wherein the 5′ segment is complementary to a segment at the 5′ end of the amplified nucleic acid strand;
(c) annealing the 5′ segment of the stem-loop adaptor to the 5′ end segment of the amplified nucleic acid strand, thereby forming a partially duplex, two-stranded intermediate structure wherein the 5′ phosphate group of the amplified nucleic acid strand is separated by a nick from the 3′ hydroxyl group of the stem-loop adaptor; and
(d) providing a ligase that seals the nick between the 5′ phosphate group of the amplified nucleic acid strand and the 3′ hydroxyl group of the stem-loop adaptor and additionally links the 5′ phosphate group of the stem-loop adaptor to the 3′ hydroxyl group of the amplified nucleic acid strand, thereby forming a closed nucleic acid structure.
2. The method of claim 1 , wherein a segment of the amplified nucleic acid strand located immediately adjacent and internal to the 5′ end segment is complementary to a segment at the 3′ end of the amplified nucleic acid strand.
3. The method of claim 2 , wherein at least the 3′-most nucleobase unit of the 3′ end segment is complementary to the 5′-most nucleobase unit of the segment adjacent to the 5′ end segment.
4. The method of claim 3 , wherein the 3′ end segment of the amplified nucleic acid strand is capable of forming at least 2, 3, or 4 nucleobase unit pairs with the segment adjacent to the 5′ end segment.
5. The method of claim 1 , wherein at least a first primer of the pair includes a 5′ segment located between the 5′ phosphate group and the 3′ segment.
6. The method of claim 5 , wherein the 5′ segment of the first primer of the pair includes a sequence which is complementary to the 5′ segment of the stem-loop adaptor.
7. The method of claim 6 , wherein the first primer of the pair includes an additional segment between the 5′ and 3′ segments, wherein the additional segment is complementary to a segment at the 3′ end of the amplified nucleic acid strand.
8. The method of claim 1 , further comprising:
(e) contacting a complementary strand of the denatured amplified nucleic acid with a second stem-loop adaptor having a 5′ phosphate group, a 5′ segment, a 3′ segment having a stem-loop structure, and a 3′ hydroxyl group, wherein the 5′ segment is complementary to a segment at the 5′ end of the complementary amplified nucleic acid strand;
(f) annealing the 5′ segment of the second stem-loop adaptor to the 5′ end segment of the denatured complementary amplified nucleic acid strand, thereby forming a partially duplex, two-stranded nucleic acid structure wherein the 5′ phosphate group of the complementary amplified nucleic acid strand is separated by a nick from the 3′ hydroxyl group of the second stem-loop adaptor; and
(g) providing a ligase that seals the nick between the 5′ phosphate group of the complementary amplified nucleic acid strand and the 3′ hydroxyl group of the second stem-loop adaptor and additionally links the 5′ phosphate group of the second stem-loop adaptor to the 3′ hydroxyl group of the complementary amplified nucleic acid strand, thereby forming a closed nucleic acid structure.
9. The method of claim 8 , wherein a segment of the complementary amplified nucleic acid strand located immediately adjacent and internal to the 5′ end segment is complementary to a segment at the 3′ end of the complementary amplified nucleic acid strand.
10. The method of claim 8 , wherein the second primer of the pair includes a 5′ segment located between the 5′ phosphate group and the 3′ segment.
11. The method of claim 10 , wherein the 5′ segment of the second primer of the pair includes a sequence tag which is complementary to the 5′ segment of the second stem-loop adaptor.
12. The method of claim 11 , wherein the second primer of the pair includes an additional segment between the 5′ and 3′ segments, wherein the additional segment is complementary to a segment at the 3′ end of the complementary amplified nucleic acid strand.
13. The method of claim 8 , wherein steps (e), (f), and (g) are performed in parallel or in series with steps (b), (c), and (d).
14. The method of claim 8 , wherein steps (e), (f), and (g) are performed in reaction mixtures which are the same reaction mixtures in which with steps (b), (c), and (d), respectively, are performed.
15. A method of forming a closed nucleic acid structure comprising a segment of a target nucleic acid, the method comprising:
(a) contacting the target nucleic acid with a primer having a 5′ phosphate group and a 3′ segment, the 3′ segment being a target-binding segment;
(b) annealing the 3′ segment of the primer to the target nucleic acid, thereby forming a template for extension from the primer;
(c) forming an extended nucleic acid strand duplexed to the target nucleic acid, the extended strand comprising from 5′-3′ the primer and a segment complementary to the target nucleic acid, the 5′ end of the extended strand being the phosphorylated 5′ end of the primer and the 3′ end of the extended strand having a 3′ hydroxyl group;
(d) denaturing the extended strand from the template nucleic acid and contacting the extended strand with a stem-loop adaptor having a 5′ phosphate group, a 5′ segment, a 3′ segment having a stem-loop structure, and a 3′ hydroxyl group, wherein the 5′ segment is complementary to a segment at the 5′ end of the extended strand;
(e) annealing the 5′ segment of the stem-loop adaptor to the 5′ segment of the extended strand, thereby forming a partially duplex, two-stranded intermediate structure wherein the 5′ phosphate group of the extended strand is separated by a nick from the 3′ hydroxyl group of the stem-loop adaptor; and
(f) providing a ligase that seals the nick between the 5′ phosphate group of the extended strand and the 3′ hydroxyl group of the stem-loop adaptor and additionally links the 5′ phosphate group of the stem-loop adaptor to the 3′ hydroxyl group of the extended strand, thereby forming a closed nucleic acid structure.
16. The method of claim 15 , wherein a segment of the extended strand located immediately adjacent and internal to the 5′ end segment is complementary to a segment at the 3′ end of the extended strand.
17. The method of claim 16 , wherein at least the 3′-most nucleobase unit of the 3′ end segment is complementary to the 5′-most nucleobase unit of the segment adjacent to the 5′ end segment.
18. The method of claim 15 , wherein the 5′ segment of the primer includes a sequence which is complementary to the 5′ segment of the stem-loop adaptor.
19. The method of claim 18 , wherein the primer includes an additional segment between the 5′ and 3′ segments, wherein the additional segment is complementary to a segment at the 3′ end of the extended strand.
20. A method of forming a closed nucleic acid structure comprising a segment of a target nucleic acid, the method comprising:
(a) contacting the target nucleic acid with a primer having a 5′ phosphate group and a 3′ segment, the 3′ segment being a target-binding segment;
(b) annealing the 3′ segment of the primer to the target nucleic acid, thereby forming a template for extension from the primer;
(c) forming an extended nucleic acid strand duplexed to the target nucleic acid, the extended strand comprising from 5′-3′ the primer and a strand complementary to the target nucleic acid, the 5′ end of the extended strand being the phosphorylated 5′ end of the primer and the 3′ end of the extended strand having a 3′ hydroxyl group;
(d) denaturing the extended strand from the target nucleic acid and contacting the extended strand with a stem-loop adaptor having a 5′ phosphate group, a 5′ segment having a stem-loop structure, a 3′ segment, and a 3′ hydroxyl group, wherein the 3′ segment is complementary to a segment located at the 3′ end of the extended strand;
(e) annealing the 3′ segment of the stem-loop adaptor to the 3′ end segment of the extended strand, thereby forming a partially duplex, two-stranded intermediate structure wherein the 5′ phosphate group stem-loop adaptor is separated by a nick from the 3′ hydroxyl group of the extended strand; and
(f) providing a ligase that seals the nick between the 5′ phosphate group of the extended strand and the 3′ hydroxyl group of the stem-loop adaptor and additionally links the 5′ phosphate group of the stem-loop adaptor to the 3′ hydroxyl group of the extended strand, thereby forming a closed nucleic acid structure.