Engineering DNA assembly in vivo and methods of making and using the reverse transcriptase technology

Patent metadata

Field	Value
Publication number	US-9970040-B2
Application number	US-201414498116-A
Country	US
Kind code	B2
Filing date	Sep 26, 2014
Priority date	Sep 26, 2013
Publication date	May 15, 2018
Grant date	May 15, 2018

Abstract

Cells that can synthesize oligonucleotides in vivo to produce a nucleic acid nanostructure are described. Methods for producing oligonucleotide nanostructures for use in regulating gene expression and altering biological pathways are provided. Methods of performing multiplex automated genome editing (MAGE) are also provided.

First claim

What is claimed is:

1. A method for synthesizing a single stranded DNA (ssDNA) oligonucleotide in a cell, comprising: expressing a reverse transcriptase in the cell and expressing in the cell a functional template comprising a tRNA structure is fused to a terminator or 3′ end of a non-coding RNA sequence, wherein the tRNA structure is capable of initiating transcription of the non-coding RNA sequence using the reverse transcriptase to produce the ssDNA oligonucleotide in the cell, wherein the cell is a bacteria, wherein the bacteria lacks intracellular exonuclease, and wherein the ssDNA oligonucleotide is 8-200 nucleotides in length.

2. A method for synthesizing a single stranded DNA (ssDNA) oligonucleotide in a cell, comprising: expressing a reverse transcriptase in the cell and expressing in the cell a functional template comprising a tRNA structure is fused to a terminator or 3′ end of a non-coding RNA sequence, wherein the tRNA structure is capable of initiating transcription of the coding RNA sequence using the reverse transcriptase to produce the ssDNA oligonucleotide in the cell, wherein the cell lacks intracellular exonuclease, further comprising expressing a second reverse transcriptase in the cell.

3. The method of claim 2 , wherein the second reverse transcriptase is MLRT.

4. The method of claim 1 , wherein the reverse transcriptase is HIVRT.

5. The method of claim 4 , wherein the HIVRT comprises p66 linked to p51.

6. The method of claim 5 , wherein the p66 domain includes an N-terminal finger, palm and thumb domain.

7. The method of claim 1 , wherein the tRNA structure is tRNA Lys .

8. The method of claim 1 , wherein the ssDNA oligonucleotide includes deoxyribonucleotides and ribonucleotides.

9. The method of claim 1 , further comprising isolating the ssDNA oligonucleotide from the cell.

10. The method of claim 9 , wherein ssDNA oligonucleotide is processed to remove the ribonucleotides.

11. The method of claim 1 , wherein the ssDNA oligonucleotide includes only deoxyribonucleotides.

12. The method of claim 1 , wherein the ssDNA oligonucleotide is used in the synthesis of a nanostructure.

13. The method of claim 1 , further comprising using the ssDNA oligonucleotide in a method of DNA origami.

14. The method of claim 1 , wherein the ssDNA oligonucleotide is 10-100 nucleotides in length.

15. The method of claim 1 , wherein the reverse transcriptase is expressed under the control of an inducible promoter.

Assignees

• Massachusetts Inst Technology

Inventors

• Elbaz Johann
• Voigt Christopher

Classifications

• C07H21/00

  Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids · CPC title

• C12N15/1096

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• C12N9/1276

  RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title