ONE POT ASSEMBLY

• US 20190322998A1
• Filed: 04/27/2017
• Published: 10/24/2019
• Est. Priority Date: 04/27/2017
• Status: Abandoned Application

• Current Assignee: University of Southampton
• Original Assignee: University of Southampton
• Inventors: Tavassoli, Ali
• Application Number:
  US15/499,841
• Publication Number:
  US 20190322998A1
• Time in Patent Office: Days
• CPC Class Codes: 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
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR

Abstract

Disclosed herein are methods of synthesising a double stranded polynucleotide, the methods including providing a set of self-templating oligonucleotides wherein the set of self-templating oligonucleotides encodes a double stranded polynucleotide sequence of interest; annealing the oligonucleotides together so that they form the double stranded polynucleotide sequence with the oligonucleotides in the correct order and adjacent oligonucleotides in the same strand close enough to each other that a covalent bond could form between the 5′ end of one oligonucleotide and the 3′ end of an adjacent oligonucleotide; and covalently bonding the backbones of adjacent oligonucleotides in the same strand to each other so that a covalent bond is formed between the 5′end of an oligonucleotide to the 3′end of the adjacent oligonucleotide to provide the double stranded polynucleotide of interest, wherein the covalent bonds between adjacent oligonucleotides can be read-through accurately by a DNA polymerase or an RNA polymerase.

Claims

1. A method of synthesising a double stranded polynucleotide comprising:

providing a set of self-templating oligonucleotides wherein the set of self-templating oligonucleotides encodes a double stranded polynucleotide sequence of interest;

annealing the oligonucleotides together so that they form the double stranded polynucleotide sequence with the oligonucleotides in the correct order and adjacent oligonucleotides in the same strand close enough to each other that a covalent bond could form between the 5′

end of one oligonucleotide and the 3′

end of an adjacent oligonucleotide; and

covalently bonding the backbones of adjacent oligonucleotides in the same strand to each other so that a covalent bond is formed between the 5′

end of an oligonucleotide to the 3′

end of the adjacent oligonucleotide to provide the double stranded polynucleotide of interest, wherein the covalent bonds between adjacent oligonucleotides can be read-through accurately by a DNA polymerase or an RNA polymerase.

2. The method according to claim 1, wherein the 5′

and/or 3′

ends of adjacent oligonucleotides are functionalised to enable a specific and selective chemical reaction to form a covalent bond between a functionalised group on the 5′

end of an oligonucleotide and a functionalised group on the 3′

end of an adjacent oligonucleotide.

3. The method according to claim 1, wherein the 5′

and/or 3′

ends of adjacent oligonucleotides are functionalised to enable formation of disulphide bonds, amide bonds, alkene bonds, alkane bonds, bonds between any two heteroatoms joined together or bonds between any heteroatom joined to carbon.

4. The method according to claim 1, wherein the set of oligonucleotides comprises at least two oligonucleotides encoding the sense strand of the DNA and at least two oligonucleotides encoding the antisense strand of the DNA.

5. The method according to claim 1, wherein the set of oligonucleotides covers the whole sequence of the double stranded DNA.

6. The method according to claim 1, wherein the chemical reaction between the functionalized termini is initiated by addition of a catalyst.

7. The method according to claim 1, wherein the catalyst is not an enzyme, for example, not a ligase.

8. The method according to claim 1, wherein the catalyst is Cu (I).

9. The method according to claim 1, wherein the covalent bonds between each oligonucleotide can be read through by DNA and/or RNA polymerase enzymes, Prokaryotic and/or Eukaryotic transcription factors, and DNA and/or RNA replication machinery in vivo.

10. The method according to claim 1, wherein all of the oligonucleotides in the set are annealed together in a single reaction.

11. The method according to claim 1, wherein the double stranded DNA comprises a gene.

12. The method according to claim 1, wherein the double stranded DNA is at least 300 base pairs in length.

13. The method according to claim 1, wherein each of the oligonucleotides is at least 70 base pairs in length.

14. The method according to claim 1, wherein at least one of the oligonucleotides in the set of oligonucleotides is a chemically synthesised oligonucleotide.

15. The method according to claim 1, wherein all of the oligonucleotides in the set are chemically synthesised oligonucleotides.

16. The method according to claim 1, wherein at least one of the oligonucleotides comprises at least one epigenetic modification.

17. The method according to claim 1, wherein at least one of the oligonucleotides comprises at least one methylated base.

18. The method according to claim 1, wherein at least one of the oligonucleotides comprises at least one modified or non-natural nucleotide.

19. The method according to claim 1, wherein the oligonucleotides are covalently joined together without using a ligase.

20. The method according to claim 1, wherein the 5′

end of an oligonucleotide is functionalised with an alkyne group, the 3′

end of an adjacent oligonucleotide is functionalised with an azide group, and the covalent bond formed between the two functionalised groups is a triazole phosphodiester mimic.

21. The method according to claim 1, wherein the method further comprises the steps of:

i) ligating the double stranded polynucleotide into a vector, preferably an expression vector; and

ii) transforming cells with the expression vector such that the cells express the open reading frame.

22. A double stranded DNA sequence made by the method of claim 1.

23. An expression vector comprising a DNA sequence made by the method of claim 1.

24. A cell comprising a DNA sequence made by the method of claim 1.

25. A cell comprising an expression vector comprising a sequence made by the method of claim 1.