5′ adapter comprising an internal 5′-5′ linkage
This disclosure provides, among other things, a 5′ adapter of the formula 3′*—X—(5′5′)—Y—3′, where: 3′* is a blocked 3′ end, X is a synthetic sequence, (5′5′) is an internal 5′-5′ linkage, Y is an adapter sequence, and 3′ is a hydroxylated 3′ end. In use, sequence X hybridizes to sequence X′ in a population of RNA molecules of formula R—X′, which increases the efficiency of ligation of the 5′ adapter to the nucleic acid molecules.
1. A 5′ adapter of the formula
3′*—X—(5′5′)—Y—3′,
wherein: 3′* is a blocked 3′ end, X is a sequence of at least 8 nucleotides, (5'5′) is an internal 5′-5′ linkage; Y is an adapter sequence of at least 8 nucleotides, X and Y are not complementary, and 3′ is a hydroxylated 3′ end.
2. The 5′ adapter of claim 1 ,
wherein: (i) X is complementary to sequence X′, and wherein X′ comprises a homopolymeric sequence, or (ii) X is complementary to sequence X′, and wherein X′ is comprised within a 3′ adapter.
3. The 5′ adapter of claim 1 , wherein X is an anchored homopolymer of sequence (T) n VN, (U) n VN, (A) n BN, (C) n DN, or (G) n HN, wherein n is at least 6.
4. A kit comprising a 5′ adapter of claim 1 provided in a container.
5. The kit of claim 4 , further comprising a 3′ adapter comprising sequence X′.
6. The kit of claim 4 , further comprising a polymerase capable of adding sequence X′ to an RNA, wherein sequence X′ is a homopolymeric tail.
7. The kit of claim 4 , further comprising a reverse transcription primer that has a 3′ sequence and optionally, a 5′ tail, and wherein the 3′ sequence hybridizes to sequence X′.
8. The kit of claim 7 , wherein the 3′ sequence that hybridizes to sequence X′ hybridizes to poly(A), poly(G), or poly(U).
9. The kit of claim 7 , wherein the reverse transcription primer has a 3′ sequence of sequence (T) n VN, (U) n VN, (A) n BN, (C) n DN, or (G) n HN, wherein n is at least 6.
10. The kit of claim 4 , further comprising at least two PCR primers, comprising: (i) a first PCR primer, having a 3′ sequence that is complementary to the cDNA copy of sequence Y of the 5′ adapter; and (ii) a second PCR primer.
11. The kit of claim 4 , wherein the 5′ adapter is of the formula
3′*—X—(5′5′)—Y—Z—3′,
wherein Z is a random sequence of at least two nucleotides.
12. An oligonucleotide of the formula:
3′*—X—(5′5′)—Y—Z—3′,
wherein:
3′* is a blocked 3′ end,
X is a homopolymer of at least 8 nucleotides or a sequence of at least 8 nucleotides;
5′5′ is an internal 5′-5′ linkage;
Y is an adapter sequence of at least 8 nucleotides;
Z is a random sequence of at least two nucleotides;
X and Y are not complementary; and
3′ is a hydroxylated 3′ end.
13. A method for ligating a 5′ adapter to RNA, comprising:
incubating a reaction mixture comprising:
(i) a 5′ adapter of the formula
3′*—X—(5′5′)—Y—3′,
wherein: 3′* is a blocked 3′ end, X is a sequence of at least 8 nucleotides, (5′5′) is an internal 5′-5′ linkage, Y is an adapter sequence of at least 8 nucleotides, X and Y are not complementary, and 3′ is a hydroxylated 3′ end;
(ii) a population of nucleic acid molecules of the formula 5′-R—X′-3′, wherein R is 5′-phosphorylated RNA and X′ is complementary to sequence X in the 5′ adapter; and
(iii) a ligase capable of ligating a 5′ phosphate to a 3′ hydroxyl,
under conditions by which sequence X of the 5′ adapter hybridizes to sequence X′ of the nucleic acid molecules to produce complexes and, in the complexes, the hydroxylated 3′ end of the 5′ adapter ligates to the 5′ end of the nucleic acid molecules to produce product molecules of formula 3′*—X—(5′5′)—Y—R—X′-3′.
14. The method of claim 13 , wherein sequence X′ in the nucleic acid molecules is a homopolymeric tail.
15. The method of claim 13 , wherein sequence X′ in the nucleic acid molecules is a 3′ adapter that has been ligated to the RNA.
16. The method of claim 13 , wherein the population of nucleic acid molecules of (ii) is made by: i. exposing an RNA sample to fragmentation conditions to produce RNA fragments; and ii. adding sequence X′ onto the 3′ end of the RNA fragments.
17. The method of claim 13 , further comprising:
reverse transcribing the product molecules to produce cDNA of formula 3′—Y′—R′—X—5′, wherein the internal 5′-5′ linkage blocks reverse transcription of sequence X of the product molecules.
18. The method of claim 13 , wherein the population of nucleic acid molecules of (ii) comprises: i. small RNAs and RNA fragments; and ii. sequence X′, wherein the cDNA comprises cDNA copies of the small RNAs and cDNA copies of the RNA fragments.
19. The method of claim 17 , further comprising amplifying the cDNA.
20. The method of claim 17 , further comprising sequencing the cDNA, or an amplification product thereof.
21. The method of claim 13 , wherein the 5′ adapter of (i) is of the formula
3′*—X—(5′5′)—Y—Z—3′,
wherein Z is a random sequence of at least two nucleotides.