# US 2023/0348945 A1 · Universal template strands for enzymatic polynucleotide synthesis

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- Number: US 2023/0348945 A1
- Document ID: US20230348945A1
- Status: Published application
- Kind: A1
- Application number: 18/219,079
- Filed: 2023-07-06
- Published: 2023-11-02
- Earliest claimed: 2020-05-01
- Assignees: Microsoft Technology Licensing, LLC
- Applicants: Microsoft Technology Licensing, LLC
- Inventors: Bichlien NGUYEN; Jake SMITH; Robert CARLSON; Karin STRAUSS
- Web page: https://patentgrep.com/patent/US20230348945A1

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Y 207/07007: DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12Q 1/68: involving nucleic acids
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 2521/101: DNA polymerase
- C12Q 2521/131: Terminal transferase
- C12Q 2525/101: incorporating non-naturally occurring nucleotides, e.g. inosine
- C12Q 2525/186: incorporating a non-extendable or blocking moiety
- C12Q 2533/101: Primer extension
- 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)}
- C12Y: ENZYMES
- 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

## Abstract

A universal template strand built with universal base analogs is used as a template for polynucleotide synthesis. The universal template strand can hybridize to any sequence of nucleotides. A new polynucleotide is synthesized by using a polymerase to extend a primer hybridized to the universal template strand. Unlike primer extension in polymerase chain reactions, base pairing with nucleotides in the template strand does not specify the sequence of the new polynucleotide. Instead, the sequence of the new polynucleotide is specified by the order of addition of protected nucleotides. After addition of a single species of protected nucleotide, the blocking group is removed and another protected nucleotide is added. The order of nucleotide addition can be varied to create any sequence. After synthesis, the polynucleotide can be dehybridized from the universal template strand. The universal template strand may then be reused to synthesize a different polynucleotide.

## Claims

### Claim 1

A method of enzymatic synthesis of a polynucleotide, the method comprising:
a. contacting a first primer region of a universal template strand comprising universal base analogs with a complementary primer;
b. removing a blocking group;
c. contacting the universal template strand with a protected nucleotide selected according to a predetermined polynucleotide sequence and a polymerase so that the protected nucleotide is incorporated into the polynucleotide hybridized to the universal template strand, wherein the polymerase is terminal deoxynucleotidyl transferase (TdT); and
d. repeating steps b-c to synthesize the polynucleotide.

### Claim 2 (depends on 1)

The method of claim 1, wherein the universal template strand comprises a universal region consisting of a mixture of natural bases and the universal base analogs.

### Claim 3 (depends on 2)

The method of claim 2, wherein the natural bases are present at regular intervals among the universal base analogs.

### Claim 4 (depends on 1)

The method of claim 1, wherein the universal base analogs comprise hydrogen bonding bases that form hydrogen bonds with any natural nucleobases.

### Claim 5 (depends on 1)

The method of claim 1, wherein the universal base analogs consist of inosine and derivative thereof.

### Claim 6 (depends on 1)

The method of claim 1, wherein the complementary primer includes the blocking group.

### Claim 7 (depends on 1)

The method of claim 1, wherein the blocking group is a 3′ blocking group.

### Claim 8 (depends on 1)

The method of claim 1, wherein the complementary primer has a Tm that is greater than 60° C.

### Claim 9 (depends on 1)

The method of claim 1, wherein the blocking group is thermolabile, acid-labile, redox-labile, or photolabile.

### Claim 10 (depends on 1)

The method of claim 1, wherein the polymerase is a DNA-dependent DNA polymerase.

### Claim 11 (depends on 1)

The method of claim 1, wherein a backbone of the universal template strand comprises peptide nucleic acids, bridged nucleic acids, locked nucleic acids, or ribose phosphate with a 2′-deoxy substitution.

### Claim 12 (depends on 1)

The method of claim 1, further comprising:
determining that synthesis of the predetermined polynucleotide sequence of the polynucleotide is complete; and
dehybrizing the polynucleotide from the universal template strand.

### Claim 13 (depends on 1)

The method of claim 1, wherein the universal template strand further comprises a second primer region and the method further comprises contacting the universal template strand with a mixture of nucleotides without blocking groups.

### Claim 14

A method of synthesizing a plurality of polynucleotides having different, predetermined sequences, the method comprising:
a. contacting primer regions of a plurality of universal template strands comprising universal base analogs with complementary primers, wherein the universal template strands are bound to a solid substrate;
b. removing blocking groups from nucleotides hybridized to a subset of the universal template strands;
c. contacting the plurality of universal template strands with a protected nucleotide selected according to a predetermined polynucleotide sequence and a polymerase so that the protected nucleotide is incorporated into polynucleotides hybridized to the subset of the universal template strands, wherein the polymerase is terminal deoxynucleotidyl transferase (TdT); and
d. repeating steps b-c with variations in the subset of the universal template strands and in a base of the protected nucleotide to synthesize the plurality of polynucleotides having different, predetermined sequences.

### Claim 15 (depends on 14)

The method of claim 14, wherein the solid substrate comprises a microelectrode array and removing the blocking groups comprises activating a subset of electrodes in the microelectrode array.

### Claim 16 (depends on 15)

The method of claim 15, wherein the blocking groups are removed by a redox reaction.

### Claim 17

A system for synthesizing a plurality of polynucleotides having different, predetermined sequences, the system comprising:
a solid substrate coated with a plurality of universal template strands comprising universal base analogs;
a reaction chamber containing the solid substrate;
a plurality of fluid delivery pathways each configured to introduce a single species of protected nucleotide into the reaction chamber;
a fluid delivery pathway configured to introduce a polymerase into the reaction chamber, wherein the polymerase is terminal deoxynucleotidyl transferase (TdT); and
control circuitry configured to selectively change local conditions on a portion of the surface of the solid substrate resulting in cleavage of blocking groups attached to the protected nucleotides and to selectively open the plurality of fluid delivery pathways to introduce protected nucleotides into the reaction chamber according to a predetermined polynucleotide sequence.

### Claim 18 (depends on 17)

The system of claim 17, wherein the solid substrate comprises a microelectrode array and the control circuitry is configured to selectively activate electrodes in the microelectrode array.

### Claim 19 (depends on 17)

The system of claim 17, wherein the universal template strands comprise primer regions and the system further comprises a fluid delivery pathway configured to introduce complementary primers each having a blocking group into the reaction chamber.

### Claim 20 (depends on 17)

The system of claim 17, further comprising a fluid delivery pathway configured to introduce a nucleotide mixture into the reaction chamber, wherein the new quit had mixture comprises a mixture of nucleotides having two, three, or all four natural bases.