METHODS OF NUCLEIC ACID SYNTHESIS
The invention relates to improved methods of enzymatic solid-supported nucleic acid synthesis that make use of terminal deoxynucleotidyl transferase (TdT) enzymes or modified terminal deoxynucleotidyl transferase (TdT) enzymes on polyacrylamide type supports. The invention further relates to the use of kits comprising said enzymes in a method of solid-supported nucleic acid synthesis.
1 . A method of nucleic acid synthesis, wherein the method comprises the steps of:
(a1) providing a solid support comprising particles coated with a pre-polymerised material, wherein the pre-polymerised material comprises a co-polymer to which an initiator oligonucleotide is to be attached, wherein the co-polymer is a co-polymer of one or more first co-monomer(s) selected from acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, N-(hydroxylmethyl)acrylamide, N-(hydroxyethyl)acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, hydroxyethyl methacrylate and N-vinyl pyrrolidinone and a second co-monomer which attaches to the initiator;
(a2) coupling an initiator oligonucleotide to said co-polymer to form a solid-supported initiator oligonucleotide;
(b) adding a 3ʹ-blocked nucleoside triphosphate to said initiator oligonucleotide in the presence of a terminal deoxynucleotidyl transferase (TdT) enzyme or modified terminal deoxynucleotidyl transferase (TdT) enzyme;
(c) cleaving the blocking group from the 3ʹ-blocked nucleoside triphosphate in the presence of a cleaving agent; and
(d) repeating steps (b) and (c) to synthesise an extended nucleic acid.
2 . The method according to claim 1 , wherein the first co-monomer is acrylamide.
3 . The method according to any one of claims 1 or 2 , wherein the solid support is coated with a pre-polymerised material comprising a monomer selected from acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, N-(hydroxylmethyl)acrylamide, N-(hydroxyethyl)acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, hydroxyethyl methacrylate and N-vinyl pyrrolidinone and a second co-monomer containing a reactive moiety selected from haloacetamide, carboxylic acid, alkyne, azide, amine or thiol.
4 . The method according to claim 3 , wherein the second co-monomer is a haloacetamide-containing monomer.
5 . The method according to any one of claims 1 to 4 , wherein the second co-monomer is a monomer of formula (3) or (3a):
wherein X is Cl, Br or I;
Y is NR 1 or 0;
Z is an optionally substituted C 1-50 alkyl bridge;
and R 1 is H or an optionally substituted C 1-5 alkyl group.
6 . The method according to claim 5 , wherein X is Br.
7 . The method according to claim 5 or 6 , wherein Y is NH.
8 . The method according to any one of claims 5 to 7 , wherein Z is a C5 alkyl bridge.
9 . The method according to any one of claims 1 to 8 , wherein the initiator oligonucleotide is coupled via a phosphorothioate moiety.
10 . The method according to any one of claims 1 to 8 , wherein the initiator oligonucleotide is coupled via click chemistry between an azide and an alkyne.
11 . The method according to any one of claims 1 to 10 , wherein the extended nucleic acid is detached from the solid support.
12 . The method according to any one of claims 1 to 11 , wherein the initiator contains a uracil moiety and nucleic acid is detached by removing the uracil base and cleaving the abasic site.
13 . The method according to any one of claims 1 to 12 , wherein the particles are magnetic.
14 . The method according to any one of claims 1 to 13 , wherein the particles are silica coated.
15 . The method as defined in any one of claims 1 to 14 , wherein the 3ʹ-blocked nucleoside triphosphate is blocked a group selected from 3ʹ-O-azidomethyl, 3ʹ-aminooxy, 3ʹ-O-allyl, 3ʹ-O-cyanoethyl, 3ʹ-O-acetyl, 3ʹ-O-nitrate, 3ʹ-O-phosphate, 3ʹO-acetyl levulinic ester, 3ʹ-O-tert butyl dimethyl silane, 3ʹ-O-trimethyl(silyl)ethoxymethyl, 3ʹ-O-ortho-nitrobenzyl, or 3ʹ-O-para-nitrobenzyl.
16 . The method according to any one of claims 1 to 15 , wherein the cleaving agent is tris(2-carboxyethyl)phosphine (TCEP), a palladium complex, an organic or inorganic base, sodium nitrite or a photoactivated transition metal complex.
17 . The method according to any one preceding claim , wherein the solid support contains a plurality of coagulated magnetic particles.
18 . The method according to any one preceding claim , wherein the pre-polymerisation is carried out for at least 90 minutes prior to exposure to the surface being coated.
19 . A kit comprising:
(i) a solid support comprising particles coated with a pre-polymerised material, wherein the pre-polymerised material comprises a co-polymer to which an initiator oligonucleotide is attached or is to be attached, wherein the co-polymer is a co-polymer of one or more first co-monomer(s) selected from acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, N-(hydroxylmethyl)acrylamide, N-(hydroxyethyl)acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, hydroxyethyl methacrylate and N-vinyl pyrrolidinone and a second co-monomer which attaches to or is attached to the initiator;
(ii) a 3ʹ-blocked nucleoside triphosphate;
(iii) a terminal deoxynucleotidyl transferase (TdT) enzyme or modified terminal deoxynucleotidyl transferase (TdT) enzyme and optionally;
(iv) a cleaving agent.
20 . The kit according to claim 19 further comprising an initiator oligonucleotide.
21 . The kit according to claim 19 , wherein the initiator oligonucleotide is attached to the coated support.