# US 11,993,801 B2 · Base-modified nucleotides as substrates for TdT-based enzymatic nucleic acid synthesis

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- Number: US 11,993,801 B2
- Document ID: US11993801B2
- Status: Granted
- Kind: B2
- Application number: 17/381,024
- Filed: 2021-07-20
- Published: 2022-01-27
- Granted: 2024-05-28
- Earliest claimed: 2020-07-21
- Assignees: Illumina Singapore Pte. Ltd.; Nanyang Technological University
- Applicants: Illumina Singapore Pte. Ltd.; Nanyang Technological University
- Inventors: Pin Koon Ee; Yin Nah Teo; Shunsuke Chiba
- Web page: https://patentgrep.com/patent/US11993801B2

## CPC

- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C07H 19/20: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C12N 9/1264: {DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal nucleotidyl transferase}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Y 207/07031: DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal deoxynucleotidyl transferase
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES

## Abstract

Disclosed herein include methods and compositions for nucleic acid synthesis using a terminal deoxynucleotidyl transferase with deoxyribonucleotide trisphosphates each comprising a modified base with a photocleavable carbon chain moiety that enables single incorporations when present.

## Claims

### Claim 1

A method of nucleic acid synthesis comprising:
(a) providing a nucleic acid and a plurality of nucleoside triphosphates, wherein each of the plurality of nucleoside triphosphates comprises a modified base comprising a photocleavable carbon chain moiety having a length of at least 60 Å;
(b1) contacting (i) the nucleic acid and (ii) a first nucleoside triphosphate of the plurality of nucleoside triphosphates with a first terminal deoxynucleotidyl transferase (TdT) to generate a first modified nucleic acid comprising the nucleic acid incorporated with one first nucleotide comprising the modified base from the first nucleoside triphosphate;
(c1) photocleaving the photocleavable carbon chain moiety of the modified base of the first nucleotide in the first modified nucleic acid to remove the photocleavable carbon chain moiety from the first modified nucleic acid;
(b2) contacting (i) the first modified nucleic acid with the photocleavable carbon chain moiety of the modified base of the first nucleotide removed and (ii) a second nucleoside triphosphate of the plurality of nucleoside triphosphates with a second TdT to generate a second modified nucleic acid comprising the first modified nucleic acid incorporated with one second nucleotide comprising the modified base from the second nucleoside triphosphate; and
(c2) photocleaving the photocleavable carbon chain moiety of the modified base of the second nucleotide in the second modified nucleic acid to remove the photocleavable carbon chain moiety from the second modified nucleic acid.

### Claim 2 (depends on 1)

The method of claim 1, wherein the nucleic acid is attached to a solid support, the method further comprising: detaching the modified nucleic acid from the solid support.

### Claim 3 (depends on 1)

The method of claim 1, wherein the modified base comprises a propargylamino group, an aminoallyl group, a propargylhydroxyl group or a combination thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein the photocleavable carbon chain moiety comprises a saturated or unsaturated, substituted or unsubstituted, straight or branched carbon chain, and wherein the carbon chain has a length of at least 60 Å.

### Claim 5 (depends on 1)

The method of claim 1, wherein the photocleavable carbon chain moiety comprises a plurality of repeat units.

### Claim 6 (depends on 5)

The method of claim 5, wherein the plurality of repeating units comprises a polyethylene glycol (PEG).

### Claim 7 (depends on 5)

The method of claim 5, wherein a number of the plurality of repeating units is at least 18.

### Claim 8 (depends on 1)

The method of claim 1, wherein the photocleavable carbon chain moiety comprises an photocleavable moiety selected from a group consisting of a carbonyl group, an arylcarbonylmethyl group, a phenacyl group, an o-alkylphenacyl group, a p-hydroxyphenacyl group, a benzoin group, a benzyl group, a nitroaryl group, a nitrobenzyl group, an o-nitrobenzyl group, an o-nitro-2-phenethyloxycarbonyl group, an o-nitroanilide, a coumarin-4-ylmethyl group, an arylmethyl group, a coumaryl group, an o-hydroxyarylmethyl group, a metal-containing group, a pivaloyl group, an ester of a carboxylic acid, an arylsulfonyl group, a ketone group, a carbanion-mediated group, a sisyl group, a silicon-based group, a 2-hydroxycinnamyl group, an α-keto amide group, an α,β-unsaturated anilide, a methyl(phenyl)thiocarbamic acid group, a thiochromone S,S-dioxide group, 2-pyrrolidino-1,4-benzoquinone group, a triazine group, an arylmethyleneimino group, a xanthene group, a pyronin group, a 7-hydroxy-1,1-dimethylnaphthalenone group, a carboxylic acid group, a phosphate group, a phosphite group, a sulfate group, an acid group, an alcohol group, a thiol group, a N-oxide group, a phenol group, an amine group, a derivative of any of the proceeding, or a combination thereof.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first TdT and/or the second TdT comprises a recombinant TdT.

### Claim 10 (depends on 1)

The method of claim 1, further comprising: removing or inactivating the first TdT after the step (b1) and before the step (c1); and removing or inactivating the second TdT after the step (b2) and before the step (c2).

### Claim 11 (depends on 1)

The method of claim 1,
wherein the contacting in step (b1) is performed at about 16° C. to about 58° C., and
wherein the contacting step (b2) is performed at about 16° C. to about 58° C.

### Claim 12 (depends on 1)

The method of claim 1,
wherein the first modified nucleic acid in step (b1) comprises at least 95% of the nucleic acid, and
wherein the second modified nucleic acid in step (b2) comprises at least 95% of the first modified nucleic acid.

### Claim 13 (depends on 1)

The method of claim 1,
wherein at least 95% of the first modified nucleic acid in step (b1) comprises the first modified nucleic acid comprising the nucleic acid incorporated with a single first nucleotide from the first nucleoside triphosphate, and
wherein at least 95% of the second modified nucleic acid in step (b2) comprises the second modified nucleic acid comprising the first modified nucleic acid incorporated with a single second nucleotide from the second nucleoside triphosphate.

### Claim 14 (depends on 1)

The method of claim 1,
wherein the photocleaving in step (c1) is performed with a first ultraviolet radiation, and
wherein the photocleaving in step (c2) is performed with a second ultraviolet radiation.

### Claim 15 (depends on 1)

The method of claim 1, wherein the first radiation and/or the second radiation has a wattage of about 5 watts to about 20 watts.

### Claim 16 (depends on 1)

The method of claim 1, wherein the photocleaving in the step (c1) and/or the step (c2) has an efficiency of at least 90%.

### Claim 17 (depends on 1)

The method of claim 1, wherein the contacting in step (b1) and the contacting in step (b2) each is completed in about 7 minutes.

### Claim 18 (depends on 1)

The method of claim 1, wherein the photocleaving in step (c1) and the photocleaving in step (c2) each is completed in about 1 minute.

### Claim 19 (depends on 1)

The method of claim 1, further comprising: generating a reverse complement of the modified nucleic acid using a polymerase.

### Claim 20 (depends on 1)

The method of claim 1, wherein the photocleavable carbon chain moiety comprises a polyether polymer.

### Claim 21 (depends on 20)

The method of claim 20, wherein the polyether polymer comprises paraformaldehyde, polyethylene glycol (PEG), polypropylene glycol (PPG), polyalkylene glycol (PAG), polytetramethylene glycol (PTMG), or a combination thereof.

### Claim 22 (depends on 5)

The method of claim 5, wherein a number of the plurality of repeating units is from about 18 to about 30.