Assembly and error reduction of synthetic genes from oligonucleotides

Patent metadata

Field	Value
Publication number	US-12595476-B2
Application number	US-201916982135-A
Country	US
Kind code	B2
Filing date	Mar 19, 2019
Priority date	Mar 19, 2018
Publication date	Apr 7, 2026
Grant date	Apr 7, 2026

Abstract

The present invention relates to a rapid, high-fidelity process to support synthesis of genes for in vitro transcription of modified messenger RNA. In this process, sequence errors resulting from amplification with oligonucleotides comprising inherent errors are significantly reduced.

First claim

What is claimed is:

1 . A process for reducing base pair error rate during the synthesis of a gene of interest or a fragment thereof, the process comprising the steps of:
a) obtaining a pool of assembled gene segments that contain mismatched errors, wherein the pool of assembled gene segments form the gene of interest or the fragment thereof; b) selecting error-less gene segments from the pool of assembled gene segments, comprising: i) denaturing the pool of assembled gene segments into single stranded nucleic acid sequences and forming the paired gene segments by random pairing complementary strands of the single stranded nucleic acid sequences, wherein some of the paired gene segments comprise mismatched base pairs; ii) generating a digest mixture containing error-less gene segments by mismatch digesting the paired gene segments with a T7 endonuclease I at 42° C.; iii) obtaining error-less gene segments by purifying the error-less gene segments from the digest mixture according to sizes of the error-less gene segments; and c) producing amplified error-less gene segments by amplifying the error-less gene segments using a Q5 DNA polymerase;
thereby reducing base pair error rate during the synthesis of the gene of interest or the fragment thereof, wherein the error rate of each of the amplified error-less gene segments is less than about 1 error per 4,000 base pairs synthesized.

2 . The process of claim 1 , further comprising repeating steps a) through c).

3 . The process of claim 1 , wherein the pool of assembled gene segments comprise one or more sequence errors in one or more nucleic acid sequences in the pool of assembled gene segments.

4 . The process of claim 1 , wherein the gene of interest comprises a DNA sequence or a complementary DNA sequence from a cDNA.

5 . The process of claim 1 , wherein the mismatched errors are introduced into the paired gene segment by said random pairing complementary strands of the single stranded nucleic acid sequences.

6 . The process of claim 1 , further comprising a step for determining whether the error-less gene segments have correct sizes and whether the sizes of the error-less gene segments match the size of the gene of interest or the fragment thereof.

7 . The process of claim 1 , wherein step c) is performed using polymerase chain reaction (PCR).

8 . The process of claim 1 , wherein the assembled gene segments comprise at least one exon.

9 . The process of claim 1 , wherein step a) comprises:
i) obtaining a first pool of oligonucleotides by performing at least one segmenting step on at least one nucleotide sequence comprising an open reading frame (ORF) encoding the gene of interest or the fragment thereof; ii) assembling and amplifying gene segments using the first pool of oligonucleotides and a second pool of oligonucleotides; and iii purifying the assembled and amplified gene segments produced in ii) of step a), thereby obtaining the pool of assembled gene segments.

10 . The process of claim 9 , wherein the at least one segmenting step produces gene segments with sizes up to 2000 base pairs comprising about 15-50 base pair overlap regions among the gene segments.

11 . The process of claim 10 , wherein the overlap regions comprise a GC content of about 40-60%.

12 . The process of claim 9 , wherein the first pool of oligonucleotides and the second pool of oligonucleotides are in an equimolar ratio.

13 . The process of claim 12 , wherein at least one oligonucleotide from the second pool of oligonucleotides is at least partially complementary to at least one oligonucleotide from the first pool of oligonucleotides.

14 . The process of claim 12 , wherein said assembling and amplifying gene segments comprises assembling the gene segments using a polymerase chain assembly, wherein the second pool of oligonucleotides is assembled with the first pool of oligonucleotides by the polymerase chain assembly to form the gene segments, and wherein a polymerase chain reaction (PCR) is used to amplify the gene segments.

15 . The process of claim 9 , wherein each oligonucleotide in the first pool of oligonucleotides is about 60 to 100 bases in length, and wherein each oligonucleotide in the first pool of oligonucleotides has an overlap region of about 20-50 base pairs.

16 . The process of claim 1 , wherein said purifying the error-less gene segments from the digest mixture is carried out by running the digest mixture on a gel using gel electrophoresis and recovering the error-less gene segments from the gel by gel excision after the gel electrophoresis or solid phase reversible immobilization (SPRI).

17 . The method of claim 16 , wherein the SPRI uses a buffer comprising 20% PEG8000, 2 M salt, 10 mM Tris pH 8 and 1 mM EDTA.

18 . The process of claim 1 , wherein the gene of interest or the fragment thereof comprises at least one exon sequence or at least one intron sequence.

19 . The process of claim 1 , wherein step b) comprises performing a heat denaturation reaction on the pool of assembled gene segments and slowly reducing the temperature of the heat denaturation reaction to promote said random pairing complementary strands of the single stranded nucleic acid sequences.

20 . The process of claim 1 , further comprising d) purifying the amplified error-less gene segments after step c).

21 . The process of claim 1 , further comprising e) assembling the amplified error-less gene segments into the gene of interest or the fragment thereof.

22 . A process for reducing base pair error rate during the synthesis of a gene of interest or a fragment thereof, the process comprising the steps of:
a) obtaining a pool of assembled gene segments that contain mismatched errors, wherein the assembled gene segments form the gene of interest or the fragment thereof, b) selecting error-less gene segments from the pool of assembled gene segments, comprising: i) denaturing the pool of assembled gene segments into single stranded nucleic acid sequences and forming the paired gene segments by random pairing complementary strands of the single stranded nucleic acid sequences, wherein some of the paired gene segments comprise mismatched base pairs; ii) generating a digest mixture containing error-less gene segments by mismatch digesting the paired gene segments with a T7 endonuclease I at 42° C.; iii) obtaining error-less gene segments by purifying the error-less gene segments from the digest mixture according to sizes of the error-less gene segments; and c) producing amplified error-less gene segments by amplifying the error-less gene segments using a high fidelity polymerase with proofreading activity, wherein the high fidelity polymerase with proofreading activity comprises a Q5 DNA polymerase, a Herculase DNA polymerase or a Phusion DNA polymerase, thereby reducing base pair error rate during the synthesis of the gene of interest or the fragment thereof, wherein the error rate of each of the amplified error-less gene segments is less than about 1 error per 4,000 base pairs.

23 . The process of claim 22 , comprising repeating steps a) through c).

24 . The process of claim 22 , wherein step a) comprises:
i) obtaining a first pool of oligonucleotides by performing at least one segmenting step on at least one nucleotide sequence comprising an open reading frame (ORF) encoding the gene of interest or the fragment thereof; ii) assembling and amplifying gene segments using

Assignees

• Modernatx Inc

Inventors

• Duvall Marcus R
• Smith Kevin
• Huang Robin
• Giessel Andrew

Classifications

• C12Q1/6848 Primary

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12N15/1027

  by DNA shuffling, e.g. RSR, STEP, RPR · CPC title

• C12N15/1031 Primary

  mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR · CPC title