# US 8,785,157 B2 · Method for using ref protein as a targeted reca-dependent nuclease

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- Number: US 8,785,157 B2
- Document ID: US8785157B2
- Status: Granted
- Kind: B2
- Application number: 13/208,985
- Filed: 2011-08-12
- Published: 2012-04-12
- Granted: 2014-07-22
- Earliest claimed: 2010-08-13
- Assignees: Wisconsin Alumni Research Foundation
- Applicants: Michael M. Cox; Marielle C. Eichhorn-Gruenig; James L. Keck
- Inventors: Michael M. Cox; Marielle C. Eichhorn-Gruenig; James L. Keck
- Web page: https://patentgrep.com/patent/US8785157B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C07K 14/005: from viruses
- C12N 9/00: Enzymes; Proenzymes; Compositions thereof (preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; {enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113}); Processes for preparing, activating, inhibiting, separating or purifying enzymes (preparation of malt C12C1/00)
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12Q 1/6827: for detection of mutation or polymorphism
- C12N 2310/153: with the aid of a protein, e.g. recombinase
- C12N 2795/00022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Kits and a method for cleaving double-stranded DNA using Ref and RecA protein and variants thereof at a site having a DNA sequence homologous to the sequence on a single-stranded DNA targeting fragment are disclosed.

## Claims

### Claim 1

A method of cleaving a duplex DNA molecule at any target defined nucleotide sequence, comprising the steps of:
(a) assembling a complex of a single-stranded DNA targeting fragment comprising a nucleotide sequence homologous to a target nucleotide sequence of a duplex DNA molecule with a RecA protein comprising: (i) the amino acid sequence of SEQ ID NO:1; (ii) the amino acid sequence of SEQ ID NO:1 wherein lysine is substituted for glutamic acid at amino acid residue 38, or (iii) an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:1, wherein the RecA functionality is retained;
(b) contacting the complex that is assembled in step (a) with the duplex DNA molecule; and
(c) contacting a Ref protein comprising: (i) SEQ ID NO:2, or (ii) an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:2, wherein the Ref functionality is retained with the duplex DNA molecule;
wherein both strands of the targeted duplex DNA molecule are cleaved within the sequences defined by homology to the single-stranded DNA used as the targeting fragment.

### Claim 2 (depends on 1)

The method of claim 1, wherein the RecA protein used in step 1(a) is a RecA protein comprising the amino acid sequence of SEQ ID NO:1 or a RecA protein comprising the amino acid sequence of SEQ ID NO:1 wherein lysine is substituted for glutamic acid at amino acid residue 38.

### Claim 3 (depends on 1)

The method of claim 1, wherein the Ref protein used in step 1(c) is a Ref protein comprising the amino acid sequence of SEQ ID NO:2.

### Claim 4 (depends on 1)

The method of claim 1, wherein the Ref protein comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:2 that is used in step 1(c) comprises a ββα-metal core fold motif having the amino acid sequence of amino acid residues 112-123 of SEQ ID NO:2, and further comprises two metal binding motifs, one such motif C-terminal to the ββα-metal core fold motif and comprising the amino acid sequence of amino acid residues 96-99 of SEQ ID NO:2, and one such motif N-terminal to the ββα-metal core fold motif and comprising the amino acid sequence of amino acid residues 130-133 of SEQ ID NO:2.

### Claim 5 (depends on 1)

The method of claim 1, wherein the single-stranded DNA targeting fragment is from 30-1,000 nucleotides in length.

### Claim 6 (depends on 5)

The method of claim 5, wherein the single-stranded DNA targeting fragment is from 90-1,000 nucleotides in length.

### Claim 7 (depends on 5)

The method of claim 5, wherein the nucleotide sequence on the single-stranded DNA targeting fragment that is homologous to the target nucleotide sequence is from 60-150 nucleotides in length.

### Claim 8 (depends on 1)

The method of claim 1, wherein one or more of steps 1(a), 1(b), and 1(c) occur within a solution comprising a divalent metal ion.

### Claim 9 (depends on 8)

The method of claim 8, wherein the divalent metal ion is Mg2+or Mn2+.

### Claim 10 (depends on 9)

The method of claim 9, wherein the divalent metal ion is Mg2+.