# US 2023/0053916 A1 · Methods and systems for nucleic acid synthesis

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- Number: US 2023/0053916 A1
- Document ID: US20230053916A1
- Status: Published application
- Kind: A1
- Application number: 17/550,165
- Filed: 2021-12-14
- Published: 2023-02-23
- Earliest claimed: 2020-09-01
- Assignees: Replay Therapeutics, Inc.; Neochromosome Inc.
- Applicants: Replay Therapeutics, Inc.; Neochromosome, Inc.
- Inventors: Leslie MITCHELL; Adrian WOOLFSON
- Web page: https://patentgrep.com/patent/US20230053916A1

## CPC

- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 2525/161: incorporating target specific and non-target specific sites
- 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

The present disclosure provides methods, compounds, and systems for synthesizing a nucleic acid molecule. The methods may comprise spatially separating a set of barcoded oligonucleotides corresponding to a target nucleic acid molecule and performing a nucleic acid assembly using the oligonucleotides. A computer system coupled to a process control software program may be configured to apparatuses and configured to carry out the methods of the present disclosure.

## Claims

### Claim at position 1

1-111. (canceled)

### Claim 112

A method of generating a target polynucleotide, said method comprising:
(a) providing a nucleic acid molecule comprising a stem-loop and a barcode sequence;
(b) contacting a solid support with said nucleic acid molecule, wherein said solid support comprises a capture sequence complementary to said barcode sequence, thereby forming a capture complex comprising said capture sequence and said nucleic acid molecule;
(c) separating said nucleic acid molecule from said capture complex; and
(d) incubating said nucleic acid molecule with assembly reagents, thereby generating at least a portion of said target polynucleotide.

### Claim 113 (depends on 112)

The method of claim 112, wherein said solid support comprises a bead.

### Claim 114 (depends on 112)

The method of claim 112, wherein said solid support comprises a microwell.

### Claim 115 (depends on 114)

The method of claim 114, wherein said microwell is on a printed array.

### Claim 116 (depends on 112)

The method of claim 112, wherein said nucleic acid molecule further comprises a restriction enzyme sequence.

### Claim 117 (depends on 116)

The method of claim 116, wherein said barcode sequence is positioned 5′ of said restriction enzyme sequence.

### Claim 118 (depends on 112)

The method of claim 112, wherein said assembly reagents comprise a polymerase, a ligase, a restriction enzyme, or any combination thereof.

### Claim 119 (depends on 112)

The method of claim 112, wherein said barcode sequence is interior to said stem-loop.

### Claim 120 (depends on 112)

The method of claim 112, wherein said barcode sequence is positioned 5′ of said stem-loop.

### Claim 121 (depends on 120)

The method of claim 120, wherein said barcode sequence is adjacent to said stem-loop.

### Claim 122 (depends on 120)

The method of claim 120, wherein said barcode sequence is positioned at the 5′ end of said nucleic acid molecule.

### Claim 123 (depends on 112)

The method of claim 112, wherein said barcode sequence is interior to a different stem-loop.

### Claim 124 (depends on 123)

The method of claim 123, wherein said nucleic acid molecule further comprises a restriction enzyme site adjacent to said barcode sequence.

### Claim 125 (depends on 112)

The method of claim 112, wherein said nucleic acid molecule comprises a 3′ unpaired region.

### Claim 126 (depends on 112)

The method of claim 112, wherein said nucleic acid molecule is single-stranded.

### Claim 127 (depends on 112)

The method of claim 112, wherein said separating in step (c) comprises contacting said capture complex with oligonucleotides complementary to said capture sequence.

### Claim 128 (depends on 112)

The method of claim 112, wherein said separating in step (c) comprises a thermal denaturation.

### Claim 129 (depends on 112)

The method of claim 112, wherein said nucleic acid molecule in step (a) is provided in a plurality of nucleic acid molecules comprising a stem-loop and a barcode sequence.

### Claim 130 (depends on 129)

The method of claim 129, wherein said plurality of nucleic acid molecules comprises one or more different barcode sequences.

### Claim 131 (depends on 130)

The method of claim 130, wherein said plurality of nucleic acid molecules is contained in one reaction volume.

### Claim 132 (depends on 129)

The method of claim 129, wherein said plurality of nucleic acid molecules comprises at least two nucleic acid molecules comprising different barcode sequences, wherein said different barcode sequences define a plurality of subsets of said nucleic acid molecules.

### Claim 133 (depends on 112)

The method of claim 112, wherein said target polynucleotide comprises at least 100,000 nucleotides.

### Claim 134 (depends on 133)

The method of claim 133, wherein said target polynucleotide is a gene.

### Claim 135 (depends on 134)

The method of claim 134, further comprising repeating steps (a)-(d) to form a genome of an organism.

### Claim 136 (depends on 113)

The method of claim 113, wherein said incubation is performed in a droplet comprising said bead.

### Claim 137 (depends on 136)

The method of claim 136, further comprising breaking or disrupting said droplet.

### Claim 138 (depends on 112)

The method of claim 112, wherein said incubating is carried out isothermally.

### Claim 139

An oligonucleotide comprising a barcode sequence at a position, wherein said position:
is within a 5′ localized stem loop of said oligonucleotide subsequence;
is adjacent to a type IIS restriction site of said oligonucleotide; or
is within a stem loop encoded near said 5′ stem loop of said oligonucleotide.

### Claim 140 (depends on 139)

The oligonucleotide of claim 139, wherein more than one nucleic acid bases at the 3′ end of said barcode sequence comprises a mismatch with at least the last 2 bases at the 3′ end of said oligonucleotide.