Import of unnatural or modified nucleoside triphosphates into cells via nucleic acid triphosphate transporters

Patent metadata

Field	Value
Publication number	US-10513706-B2
Application number	US-201515302874-A
Country	US
Kind code	B2
Filing date	Apr 9, 2015
Priority date	Apr 9, 2014
Publication date	Dec 24, 2019
Grant date	Dec 24, 2019

Abstract

A recombinantly expressed nucleotide triphosphate transporter efficiently imports the triphosphates of unnatural nucleotides into cells, and the endogenous cellular machinery incorporates those nucleotides into cellular nucleic acids. UBPs can therefore form within the cell's nucleic acids. Moreover, neither the presence of the unnatural triphosphates nor the replication of the UBP represents a significant growth burden. The UBP is not efficiently excised by nucleic acid repair pathways, and therefore can be retained as long as the unnatural triphosphates are available in the growth medium. Thus, the resulting cell is the first organism to stably propagate an expanded genetic alphabet.

First claim

What is claimed is:

1. An isolated cell comprising:
(a) deoxyribonucleic acid (DNA) comprising at least one unnatural base pair (UBP), the at least one UBP comprising a first unnatural nucleotide and a second unnatural nucleotide; and
(b) a heterologous nucleic acid encoding a nucleoside triphosphate transporter comprising an amino acid sequence that is at least about 85% identical to SEQ ID NO: 1, wherein the isolated cell is an Escherichia coli cell, and wherein the first unnatural nucleotide and the second unnatural nucleotide each independently comprise a base selected from the group consisting of

2. The isolated cell of claim 1 , wherein the nucleoside triphosphate transporter is engineered to transport triphosphates of the first unnatural nucleotide and the second unnatural nucleotide into the cell.

3. The isolated cell of claim 1 , wherein the nucleoside triphosphate transporter comprises an amino acid sequence with at least about 90% sequence identity to SEQ ID NO: 1.

4. The isolated cell of claim 1 , wherein the first unnatural nucleotide and the second unnatural nucleotide comprise a base selected from the group consisting of

5. The isolated cell of claim 4 , wherein the first unnatural nucleotide and the second unnatural nucleotide comprise a base selected from the group consisting of

6. The isolated cell of claim 5 , wherein the first unnatural nucleotide and the second unnatural nucleotide comprise a base selected from the group consisting of

7. The isolated cell of claim 6 , wherein the first unnatural nucleotide and the second unnatural nucleotide comprise a base selected from

8. The isolated cell of claim 1 , wherein:
(a) the first unnatural nucleotide comprises a base selected from

 and
(b) the second unnatural nucleotide comprises a base selected from

9. The isolated cell of claim 8 , wherein the second unnatural nucleotide comprises a base selected from

10. The isolated cell of claim 9 , wherein the second unnatural nucleotide comprises a base selected from

11. The isolated cell of claim 8 , wherein:
(a) the first unnatural nucleotide comprises a base which is

 and
(b) the second unnatural nucleotide comprises a base selected from

12. The isolated cell of claim 11 , wherein the second unnatural nucleotide comprises a base which is

Assignees

• Scripps Research Inst

Inventors

• Romesberg Floyd E
• Malyshev Denis A

Classifications

• C12N15/8243

  involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine · CPC title

• C12P19/34 Primary

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

• C07K14/405

  from algae · CPC title

• C07K14/295

  from Chlamydiales (O) · CPC title

• C12N15/70 Primary

  Vectors or expression systems specially adapted for E. coli · CPC title