# US 2019/0169665 A1 · Gene Synthesis by Self-Assembly of Small Oligonucleotide Building Blocks

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- Number: US 2019/0169665 A1
- Document ID: US20190169665A1
- Status: Published application
- Kind: A1
- Application number: 16/023,960
- Filed: 2018-06-29
- Published: 2019-06-06
- Earliest claimed: 2015-01-22
- Assignees: Mlp Holding Aps
- Applicants: Mlp Holding Aps
- Inventors: Morten Lorentz Pedersen; Gitte Laurette Pedersen; Tanya Sharlene Kanigan
- Web page: https://patentgrep.com/patent/US20190169665A1

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12N 15/1027: {by DNA shuffling, e.g. RSR, STEP, RPR}
- C12N 15/1031: {mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR}
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- 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)

## Abstract

The invention provides a process for synthesizing genes and other long double stranded polynucleotides by assembling very short oligonucleotides into partly double stranded polynucleotides, and then connecting these partly double stranded polynucleotide subassemblies with linkers comprised of very short oligonucleotides. In one embodiment, the correct order of the polynucleotide subassemblies is coded in overhangs present at each end of the partly double stranded polynucleotide subassemblies. Linkers having a sequence complimentary to the combined overhangs connect adjacent subassemblies, which are then ligated together. In one preferred embodiment the oligos are six bases long, for which there are only 4096 different possible sequence permutations. A complete library of oligos of this size and scale can be cost-effectively synthesized and quality controlled, avoiding the typical errors and yield issues associated with phosphoramidite synthesis of longer oligos. Furthermore, the limited oligo library size supports development of a laboratory-scale gene synthesis machine.

## Claims

### Claim 1

A method for synthesizing a double stranded polynucleotide molecule having a predefined sequence, the method comprising the steps of:
i) providing at least three single stranded oligonucleotides comprising complementary nucleotide sequence parts,
ii) contacting the least three single stranded oligonucleotides provided in step i) with each other, and
iii) creating at least one phosphodiester bond between any adjacent nucleotide in the self-assembled set of single stranded oligonucleotides from step ii) to create a double stranded polynucleotide of higher molecular weight than each of the individual single stranded oligonucleotides provided in step i).

### Claim 2 (depends on 1)

Method of claim 1 comprising the further step of:
i) providing at least two double stranded polynucleotide molecules having a predefined sequence produced using the steps i) through iii) of claim 1,
ii) contacting the at least two double stranded polynucleotides provided in step i) with each other, and
iii) creating at least one phosphodiester bond between any adjacent nucleotide in the self-assembled set of double stranded polynucleotides from step ii) to create a double stranded polynucleotide of higher molecular weight than each of the individual double stranded oligonucleotides provided in step i).

### Claim 3 (depends on 1)

Method of claim 1 comprising the further step of:
i) providing at least two double stranded polynucleotide molecules having a predefined sequence produced using the steps i) through iii) of claim 1,
ii) providing at least one single stranded oligonucleotide comprising complementary nucleotide sequence parts to overhangs at the ends of the at least two double stranded polynucleotide molecules provided in step i),
iii) contacting the at least two double stranded polynucleotides provided in step i) with the at least one single stranded oligonucleotide provided in step ii), and
iv) creating at least one phosphodiester bond between any adjacent nucleotide in the self-assembled set of double stranded polynucleotides from step iii) to create a double stranded polynucleotide of higher molecular weight than each of the individual double stranded oligonucleotides provided in step i).

### Claim 4

A method for synthesizing a double stranded polynucleotide molecule having a predefined sequence, the method comprising the steps of:
i) providing at least two double stranded polynucleotide molecules having a predefined sequence,
ii) providing at least one single stranded oligonucleotide comprising complementary nucleotide sequence parts to overhangs at the ends of the at least two double stranded polynucleotide molecules provided in step i),
iii) contacting the at least two double stranded polynucleotides provided in step i) with the at least one single stranded oligonucleotide provided in step ii), and
iv) creating at least one phosphodiester bond between any adjacent nucleotide in the self-assembled set of double stranded polynucleotides from step iii) to create a double stranded polynucleotide of higher molecular weight than each of the individual double stranded oligonucleotides provided in step i).

### Claim 5 (depends on 1, 2, 3, 4)

Method of claim 1 to 4 wherein the creation of at least one phosphodiester bond is catalyzed by a ligase enzyme.

### Claim 6 (depends on 1, 2, 3, 4)

Method of claim 1 to 4 wherein the creation of at least one phosphodiester bond is substituted by combining, in a polymerase chain reaction, the individual oligonucleotides and polynucleotides into at least one double stranded polynucleotide of higher molecular weight than the each of the individual oligonucleotides/polynucleotides that went into the reaction.

### Claim 7 (depends on 2, 3, 4)

Method of claim 2 to 4 wherein each of the at least two double stranded polynucleotide molecules provided in step i) comprises no more than one 3′ overhang and no more than one 5′ overhang.

### Claim 8 (depends on 2, 3, 4)

Method of claim 2 to 4 wherein at least one of the at least two double stranded polynucleotide molecules provided in step i) is treated with a phosphotase prior to step iii).

### Claim 9 (depends on 2, 3, 4)

Method of claim 2 to 4 wherein one of the at least two double stranded polynucleotides provided in step i) is attached to a solid support.

### Claim 10 (depends on 1, 2, 3, 4)

Method of claim 1 to 4, wherein the double stranded polynucleotide is assembled by an automated process or a semi-automated process.

### Claim 11 (depends on 1, 3, 4)

Method of claim 1, 3, and 4, wherein the at least one single stranded oligonucleotide provided in step i) of claim 1 and the at least one single stranded oligonucleotide provided in step ii) of claim 3 and 4 is derives from a single stranded tag library extracted from at least one biological source.

### Claim 12 (depends on 1, 3, 4)

Method of claim 1, 3, and 4, wherein the at least one single stranded oligonucleotide provided in step i) of claim 1 and the at least one single stranded oligonucleotide provided in step ii) of claim 3 and 4 derives from a single stranded tag library extracted from at least one synthetic oligo/poly-nucleotide.

### Claim 13 (depends on 1, 3)

Method of claim 1, 3, and 4, wherein the three-dimensional structure of the resulting molecule in step iii) comprises a double-helix structure.

### Claim 14 (depends on 1, 3, 4)

Method of claim 1, 3 and 4 wherein the at least one single stranded oligonucleotide provided in step i) of claim 1 and the at least one single stranded oligonucleotide provided in step ii) of claim 3 and 4 derives from a library comprising single stranded oligonucleotides comprising all possible sequence permutations of said single stranded oligonucleotide or any fraction of all possible sequence permutations of said single stranded oligonucleotide, such as at least 90% of all possible sequence permutations of said single stranded oligonucleotide.

### Claim 15 (depends on 1)

Method of claim 1 wherein the at least three single stranded oligonucleotides with complementary nucleotide sequence parts provided step i) all have the same length.

### Claim 16 (depends on 1, 3, 4)

Method of claim 1, 3, and 4, wherein the at least one single stranded polynucleotide provided in step i) of claim 1 and step ii) of claim 3 and 4, is between 1 and 30 bases long.

### Claim 17 (depends on 4)

Method of claim 4 wherein at least one of the two double stranded polynucleotides provided in step i) is derived from a double stranded polynucleotide library extracted from at least one biological source.

### Claim 18 (depends on 4)

Method of claim 4 wherein at least one of the two double stranded polynucleotides provided in step i) is derived from a synthetic double stranded polynucleotide library.

### Claim 19 (depends on 1, 3, 4)

Method of claim 1, 3, and 4, wherein the at least one single stranded oligonucleotide provided in step i) of claim 1 and the at least one single stranded oligonucleotide provided in step ii) of claim 3 and 4 is at least one base longer than the combined length of its two complementary zip codes; providing a gap in one strand of the resulting polynucleotide assembly which can subsequently be closed by a DNA polymerase, or other method known in the art.