Modified nucleotides and nucleotide conjugates for polynucleotide synthesis
Disclosed herein are improved methods for the de novo synthesis of nucleic acids by cyclic extension using a template-independent polymerase. Secondary structure formation in the nascent chain, which may inhibit extension reactions, is suppressed by the use of monomers with O-alkylated nucleobases that prevent Watson-Crick base pairing and/or other structures. After the synthesis is completed, the nucleobases can be converted back into native form by enzymatic removal of the alkyl groups.
1 . A method of synthesizing a polynucleotide, comprising:
providing a polynucleotide comprising one or more alkylated nucleobases;
enzymatically removing one or more alkyl groups from said one or more alkylated nucleobases.
2 . The method of claim 1 , wherein providing said polynucleotide comprises:
contacting a precursor polynucleotide with a polymerase and a nucleotide comprising said alkylated nucleobase;
adding said nucleotide to the 3′ end of said precursor polynucleotide via said polymerase.
3 . The method of claim 1 , wherein providing said polynucleotide comprises:
contacting a precursor polynucleotide with a conjugate comprising a nucleotide covalently linked to a polymerase via a linker, wherein said nucleotide comprises said alkylated nucleobase; and
adding said nucleotide to the 3′ end of said precursor polynucleotide via said polymerase.
4 . The method of claim 3 , further comprising cleaving said linker after addition of said nucleotide to said precursor polynucleotide.
5 . The method of claim 2 , wherein said polymerase is a template independent polymerase.
6 . The method of claim 2 , wherein the nucleotide comprises a 2′ or 3′ modification.
7 . The method of claim 1 , wherein said removal of said one or more alkyl groups comprises contacting said polynucleotide with an enzyme capable of removing said one or more alkyl groups from said alkylated nucleobases.
8 . The method of claim 7 , wherein said enzyme is an alkyl transferase.
9 . The method of claim 3 , wherein the conjugate is represented by:
wherein
L 1 is selected from the group consisting of an optionally substituted C 1-4 alkylene chain, wherein 1-2 methylene units is optionally and independently replaced with —O—, —N(R a )—, —C(O)—, —S—, —S(O)—, —S(O) 2 —, or phenylene;
L 2 is a cleavable linker;
each R a is independently hydrogen or C 1-6 alkyl;
R 1 is C 1-6 alkyl, C 2-6 alkenyl, C 1-6 alkynyl, and —(CH 2 ) 0-3 Ph, wherein R 1 is optionally substituted with 1-6 instances of R 1a ;
each R 1a is independently selected from halogen, C 1-6 alkyl, —(CH 2 ) 0-3 OR 1b , —NO 2 , —N 3 , —OPO 2 OH, and —(CH 2 ) 0-3 NHR 1b ; and
each R 1b is independently selected from hydrogen, C 1-6 alkyl, —C(O)(C 1-6 alkyl), C 1-6 haloalkyl, —C(O)(C 1-6 haloalkyl), and —CH 2 OAc;
R is a ribose polyphosphate or deoxyribose polyphosphate; and
Pol is a polymerase.
10 . The method of claim 9 , wherein R 1 is C 1-3 alkyl.
11 . The method of claim 10 , wherein R 1 is methyl.
12 . The method of claim 9 , wherein the conjugate is