Closed nucleic acid structures

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.

1 - 22 . (canceled) 
     
     
         23 . A method of forming a closed nucleic acid structure comprising a target nucleic acid, the method comprising:
 (a) contacting a target nucleic acid with a first primer having a 3′ target-binding segment, thereby forming a template for extension from the first primer;   (b) forming a first extended nucleic acid strand duplexed to the target nucleic acid, the first extended strand comprising from 5′-3′ the first primer and a segment complementary to the target nucleic acid;   (c) denaturing the first extended strand from the target nucleic acid and contacting the first extended strand with a second primer having a 5′ phosphate group and a 3′ segment complementary to a segment of the first extended strand;   (d) annealing the 3′ segment of the second primer to the first extended strand, thereby forming a template for extension from the second primer;   (e) forming a second extended nucleic acid strand duplexed to the first extended strand, the second extended strand comprising from 5′-3′ the second primer, a segment complementary to the first extended strand and having a common sequence with the target nucleic acid, and a segment complementary to the first primer, the 5′ end of the second extended strand being the phosphorylated 5′ end of the second primer and the 3′ end of the second extended strand having a 3′ hydroxyl group;   (f) denaturing the second extended strand from the first extended strand and contacting the second extended strand with an bridging oligonucleotide having a 5′ segment and a 3′ segment, the 5′ segment being complementary to a segment at the 5′ end of the second extended strand and the 3′ segment being complementary to a segment at the 3′ end of the second extended strand;   (g) annealing the 5′ segment of the bridging oligonucleotide to the 5′ segment of the second extended strand and the 3′ segment of the bridging oligonucleotide to the 3′ segment of the second extended strand, thereby forming a partially duplex, two-stranded intermediate, wherein the 5′ phosphate group of the second extended strand is separated by a nick from the 3′ hydroxyl group of the second extended strand; and   (h) providing a ligase that seals the nick between the 5′ phosphate and 3′ hydroxyl groups of the second extended strand, thereby forming a closed nucleic acid structure.   
     
     
         24 . The method of  claim 23 , wherein the bridging oligonucleotide further comprising an additional segment between the 5′ and 3′ segments that base-pair with one or more non-template-determined nucleobase units incorporated in the second extended strand; 
     
     
         25 . The method of  claim 23 , wherein the additional segment comprises or consists of 1, 2, 3, 4, or more T nucleobase units. 
     
     
         26 . The method of  claim 23 , wherein the target nucleic acid is single-stranded. 
     
     
         27 . The method of  claim 23 , wherein the target nucleic acid is part of a duplex. 
     
     
         28 . The method of  claim 27 , wherein the first primer displaces the complementary strand of the duplex. 
     
     
         29 . The method of  claim 27 , further comprising:
 (i) contacting a strand complementary to the target nucleic acid in the target nucleic acid duplex with the second primer, thereby forming a template for extension from the second primer;   (j) forming a third extended nucleic acid strand duplexed to the strand complementary to the target nucleic acid, the third extended strand comprising from 5′-3′ the second primer and a segment having a common sequence with the target nucleic acid;   (k) denaturing the third extended strand from the strand complementary to the target nucleic acid and contacting the third extended strand with the first primer, wherein the first primer includes a 5′ phosphate group;   (l) annealing the 3′ segment of the first primer to the third extended strand, thereby forming a template for extension from the first primer;   (m) forming a fourth extended nucleic acid strand duplexed to the third extended strand, the fourth extended strand comprising from 5′-3′ the first primer, a segment complementary to the third extended strand and having a sequence complementary to the target nucleic acid, and a segment complementary to the second primer, the 5′ end of the fourth extended strand being the phosphorylated 5′ end of the first primer and the 3′ end of the fourth extended strand having a 3′ hydroxyl group;   (n) denaturing the fourth extended strand from the third extended strand and contacting the fourth extended strand with a second bridging oligonucleotide having a 5′   segment and a 3′ segment, the 5′ segment being complementary to a segment at the 5′ end of the fourth extended strand and the 3′ segment being complementary to a segment at the 3′ end of the fourth extended strand;   (o) annealing the 5′ segment of the second bridging oligonucleotide to the 5′ segment of the fourth extended strand and the 3′ segment of the second bridging oligonucleotide to the 3′ segment of the fourth extended strand, thereby forming a partially duplex, two-stranded intermediate wherein the 5′ phosphate group of the fourth extended strand is separated by a nick from the 3′ hydroxyl group of the fourth extended strand; and   (p) providing a ligase that seals the nick between the 5′ phosphate and 3′ hydroxyl groups of the fourth extended strand, thereby forming a closed nucleic acid structure.   
     
     
         30 . The method of  claim 29 , wherein steps (a), (b), (c), (d), (e), (f), (g), and (h) are performed at the same time as steps (i), (j), (k), (l), (m), (n), (o), and (p), respectively. 
     
     
         31 . The method of  claim 30 , wherein steps (a), (b), (c), (d), (e), (f), (g), and (h) are performed in reaction mixtures that are the same reaction mixtures in which steps (i), (j), (k), (l), (m), (n), (o), and (p), are respectively performed. 
     
     
         32 . The method of  claim 23 , wherein the bridging oligonucleotide is attached to a solid support. 
     
     
         33 . The method of  claim 23 , further comprising performing a sequencing reaction with the closed nucleic acid structure serving as a template for generation of a nascent strand from which sequence of the target nucleic acid is read with the sequencing reaction proceeding around the closed nucleic acid structure multiple times.