Compositions and methods for chemical cleavage and deprotection of surface-bound oligonucleotides
Embodiments of the present disclosure relate to methods of preparation of templates for polynucleotide sequencing. In particular, the disclosure relates to linearization of clustered polynucleotides in preparation for sequencing by cleavage of one or more first strands of double-stranded polynucleotides immobilized on a solid support by a transition metal complex, for example, a palladium complex or a nickel complex. Further disclosure relate to linearization of clustered polynucleotides by cleaving one or more second strands of double double-stranded polynucleotides immobilized on a solid support comprising azobenzene linker by Na 2 S 2 O 4 . Nucleotides and oligonucleotides comprising a 3′ phosphate moiety blocking group, and methods of removing the same using a fluoride reagent are also disclosed.
1. A method of linearizing a plurality of immobilized double-stranded polynucleotides, comprising:
providing a solid support comprising double-stranded polynucleotides, wherein each double-stranded polynucleotide comprises a first strand and a second strand, wherein the first strand and the second strand are immobilized to the solid support at their 5′ ends, and wherein each first strand comprises a first cleavage site capable of undergoing chemical cleavage by a transition metal complex, wherein the transition metal complex is a palladium complex;
contacting the double-stranded polynucleotides with an aqueous solution of the palladium complex, thereby cleaving one or more first strands at the first cleavage site, and generating one or more cleaved first nucleic acids and cleaved immobilized first strands; and
removing the cleaved first nucleic acids from the solid support;
wherein the first cleavage site comprises a structure of formula (II′):
wherein Base is adenine, guanine, cytosine, thymine, or uracil, or a derivative thereof.
2. The method of claim 1 , wherein each first strand is extended from a first extension primer immobilized to the solid support, and the first extension primer comprises the first cleavage site.
3. The method of claim 2 , wherein the first extension primer comprises a P5 sequence or a modified P5 sequence.
4. The method of claim 1 , wherein Base in formula (II′) is thymine.
5. The method of claim 1 , wherein the palladium complex is a palladium (0) complex.
6. The method of claim 5 , wherein the palladium (0) complex is Pd(THP) 2 , Pd(THP) 4 , Pd(THM) 4 , or combinations thereof.
7. The method of claim 1 , wherein the 3′ end of each cleaved immobilized first strand comprises a protecting group.
8. The method of claim 7 , wherein the protecting group is a 3′ end phosphate moiety having a structure of formula (I):
wherein:
R 1 is —NH 2 , —OH, —NHC(O)OR a or —OCH 2 OSi(R b ) 3 ;
R a is C 1-4 alkyl, tert-butyl, allyl, benzyl, or 9-fluorenylmethyl;
each R b is independently selected from the group consisting of C 1-4 alkyl and phenyl; and
R 2 is H, C 1-4 alkyl, an optionally substituted tetrahydrofuran, or a nucleotide.
9. The method of claim 1 , wherein each second strand is extended from a second extension primer immobilized to the solid support, and each second strand comprises a second cleavage site that is not capable of undergoing chemical cleavage by the palladium complex used for chemical cleavage of the first strand.
10. The method of claim 9 , wherein the second extension primer comprises a P7 nucleotide sequence or a modified P7 nucleotide sequence or a P17 nucleotide sequence.
11. The method of claim 9 , wherein the second cleavage site is cleaved by a method selected from the group consisting of chemical cleavage, photo cleavage, enzymatic cleavage, and a combination thereof.
12. The method of claim 11 , wherein the second cleavage site is cleaved by a chemical cleavage and wherein the second cleavage site comprises a diol linker or an azobenzene linker.
13. The method of claim 12 , wherein the diol linker comprises a structure of formula (VIII):
wherein
r is 2, 3, 4, 5, or 6; and
s is 2, 3, 4, 5, or 6.
14. The method of claim 13 , wherein the diol linker is cleavable by a periodate salt.
15. The method of claim 12 , wherein the azobenzene linker comprises a structure of Formula (X):
wherein
R 1 is H, hydroxyl, or a protected hydroxyl;
R 2 is H, C 1-6 alkyl, or C 1-6 alkoxy;
each R 3 and R 4 is independently H, halo, —C(O)OR 5 , or —C(O)NHR 6 ;
each R 5 and R 6 is independently H, C 1-6 alkyl, or C 6-10 aryl;
X is —C(O)—, —CH 2 —, or —C(O)NH—; and
each m1, m2 and m3 is independently 1, 2, 3, 4, 5, or 6.
16. The method of claim 15 , wherein the azobenzene linker is cleavable by Na 2 S 2 O 4 .
17. The method of claim 1 , wherein the double-stranded polynucleotides are immobilized to the solid support through covalent bonding.
18. The method of claim 5 , wherein Base in formula (II′) is thymine.
19. The method of claim 5 , wherein the Pd(0) complex is generated in situ from a Pd(II) complex with a water soluble phosphine.