US 2026/0226523 A1Published application

Inkjet-assisted enzymatic nucleic acid synthesis

AssigneeDNA Script

InventorsNicolas MOGHADDAM, Beatrice ADELIZZI, Tessa LOMAN, Damiano VERARDO, Daniel RODRIGUEZ-PINZON, Adrian HORGAN

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Abstract

Methods, devices, and compositions are provided for inkjet-assisted synthesis of a plurality of polynucleotides at reaction sites on a substrate using template-free polymerases, such as, terminal deoxynucleotidyl transferases (TdTs). Compositions include printable stabile formulations of synthesis reagents for inkjet delivery including, but not limited to, template-free polymerases, divalent cations, and nucleotides.

Claims20

Dependent claims

A method of enzymatically synthesizing a plurality of polynucleotides each having a predetermined sequence at reaction sites on a substrate, the method comprising:

(a) providing the substrate, wherein the substrate comprises initiators at a plurality of reaction sites, wherein each initiator has a free 3′-hydroxyl group, and wherein each polynucleotide of the plurality is assigned to a reaction site for synthesis;
(b) providing a printable reagent composition comprising a template-free polymerase, a divalent cation, a 3′-O-protected nucleoside triphosphate, and a polar organic solvent that reduces DNA melting temperature,
wherein the divalent cation:
(i) is a cobalt divalent cation (Co2+) at a concentration of about or between 0.1 mM to 1 mM, wherein the pH of the printable reagent composition is about or between 5.1 to 6.6,
(ii) is a manganese divalent cation (Mn2+) at a concentration of about or between 0.5 mM to 7 mM, wherein the pH of the printable reagent composition is about or between 6.0 to 7.0, or
(iii) is a magnesium divalent cation (Mg2+) at a concentration of about or between 1 mM to 20 mM, wherein the pH of the printable reagent composition is about or between 5.6 to 8.4;
(c) performing a reaction cycle comprising the steps of
(i) dispensing through one or more inkjet printhead nozzles at least one droplet of the printable reagent composition to each reaction site of the plurality, wherein the initiator or elongated fragments having free 3′-O-hydroxyls are reacted with the 3′-O-protected nucleoside triphosphate under suitable conditions for elongation by the template-free polymerase, wherein the initiator or elongated fragments are elongated by incorporation of the 3′-O-protected nucleoside triphosphate to form 3′-O-protected elongated fragments, and
(ii) dispensing through one or more inkjet printhead nozzles at least one droplet of a deprotection solution to deprotect the 3′-O-protected elongated fragments to form elongated fragments having free 3′-hydroxyls;
and
(d) repeating step (c) until the plurality of polynucleotides is synthesized.

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Dependencies are inferred from the claim text.

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Abstract

A method of enzymatically synthesizing a plurality of polynucleotides each having a predetermined sequence at reaction sites on a substrate, the method comprising:

(a) providing the substrate, wherein the substrate comprises initiators at a plurality of reaction sites, wherein each initiator has a free 3′-hydroxyl group, and wherein each polynucleotide of the plurality is assigned to a reaction site for synthesis;
(b) providing a printable reagent composition comprising a template-free polymerase, a divalent cation, a 3′-O-protected nucleoside triphosphate, and a polar organic solvent that reduces DNA melting temperature,
wherein the divalent cation:
(i) is a cobalt divalent cation (Co2+) at a concentration of about or between 0.1 mM to 1 mM, wherein the pH of the printable reagent composition is about or between 5.1 to 6.6,
(ii) is a manganese divalent cation (Mn2+) at a concentration of about or between 0.5 mM to 7 mM, wherein the pH of the printable reagent composition is about or between 6.0 to 7.0, or
(iii) is a magnesium divalent cation (Mg2+) at a concentration of about or between 1 mM to 20 mM, wherein the pH of the printable reagent composition is about or between 5.6 to 8.4;
(c) performing a reaction cycle comprising the steps of
(i) dispensing through one or more inkjet printhead nozzles at least one droplet of the printable reagent composition to each reaction site of the plurality, wherein the initiator or elongated fragments having free 3′-O-hydroxyls are reacted with the 3′-O-protected nucleoside triphosphate under suitable conditions for elongation by the template-free polymerase, wherein the initiator or elongated fragments are elongated by incorporation of the 3′-O-protected nucleoside triphosphate to form 3′-O-protected elongated fragments, and
(ii) dispensing through one or more inkjet printhead nozzles at least one droplet of a deprotection solution to deprotect the 3′-O-protected elongated fragments to form elongated fragments having free 3′-hydroxyls;
and
(d) repeating step (c) until the plurality of polynucleotides is synthesized.

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