# US 2019/0169240 A1 · Dna-binding protein using ppr motif, and use thereof

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- Number: US 2019/0169240 A1
- Document ID: US20190169240A1
- Status: Published application
- Kind: A1
- Application number: 16/216,617
- Filed: 2018-12-11
- Published: 2019-06-06
- Earliest claimed: 2013-04-22
- Assignees: Kyushu University, Nat'l University Corporation; Hiroshima University
- Applicants: Kyushu University, National University Corporation; Hiroshima University
- Inventors: Takashi Yamamoto; Tetsushi Sakuma; Takahiro Nakamura; Yusuke Yagi; Yasuyuki Okawa
- Web page: https://patentgrep.com/patent/US20190169240A1

## CPC

- C07K 14/415: from plants
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N 15/8217: {Gene switch}
- C12N 15/85: for animal cells
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12Y 301/21004: Type II site-specific deoxyribonuclease (3.1.21.4)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The object of the present invention is to, by analyzing PPR proteins that act to bind to DNA with a prediction that RNA recognition rules of PPR motifs can also be used for recognition of DNA, find a PPR protein showing such a characteristic. According to the present invention, it was revealed that, with a protein that can bind in a DNA base-selective manner or a DNA base sequence-specific manner, which contains one or more, preferably 2 to 30, more preferably 5 to 25, most preferably 9 to 15, of PPR motifs having a structure of the following formula 1 (wherein, in the formula 1, Helix A is a part that can form an α-helix structure; X does not exist, or is a part consisting of 1 to 9 amino acids; Helix B is a part that can form an α-helix structure; and L is a part consisting of 2 to 7 amino acids), and having a specific combination of amino acids corresponding to a DNA base or DNA base sequence as amino acids of three positions of No. 1 A.A., No. 4 A.A., in Helix A of the formula 1 and No. “ii” (−2) A.A. contained in L of the formula 1, the aforementioned object could be achieved. (Helix A)-X-(Helix B)-L (Formula 1)

## Claims

### Claim 1

A method for preparing a protein that can bind in a DNA base-selective manner or a DNA base sequence-specific manner, comprising:
providing a nucleic acid sequence encoding a protein;
cloning said nucleic acid sequence; and
preparing a transformant which produces the protein,
wherein the protein contains one or more PPR motifs having a structure of the following formula 1:
(Helix A)-X-(Helix B)-L (Formula 1)
(wherein, in the formula 1:
Helix A is a part that can form an α-helix structure;
X does not exist, or is a part consisting of 1 to 9 amino acids;
Helix B is a part that can form an α-helix structure; and
L is a part consisting of 2 to 7 amino acids),
wherein,
under the following definitions:
the first amino acid of Helix A is referred to as Number 1 amino acid (Number 1 AA), the fourth amino acid as Number 4 amino acid (Number 4 AA), and
when a next PPR motif (Mn+1) contiguously exists on the C-terminus side of the PPR motif (Mn) (when there is no amino acid insertion between the PPR motifs), the −2nd amino acid counted from the end (C-terminus side) of the amino acids constituting the PPR motif (Mn);
when a non-PPR motif consisting of 1 to 20 amino acids exists between the PPR motif (Mn) and the next PPR motif (Mn+1) on the C-terminus side, the amino acid locating upstream of the first amino acid of the next PPR motif (Mn+1) by 2 positions, i.e., the −2nd amino acid; or
when any next PPR motif (Mn+1) does not exist on the C-terminus side of the PPR motif (Mn), or 21 or more amino acids constituting a non-PPR motif exist between the PPR motif (Mn) and the next PPR motif (Mn+1) on the C-terminus side, the 2nd amino acid counted from the end (C-terminus side) of the amino acids constituting the PPR motif (Mn)
is referred to as Number “ii” (−2) amino acid (Number “ii” (−2) AA),
one PPR motif (Mn) contained in the protein is a PPR motif having a specific combination of amino acids corresponding to a target DNA base or target DNA base sequence as the three amino acids of Number 1 AA, Number 4 AA, and Number “ii” (−2) AA.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the combination of the three amino acids of Number 1 AA, Number 4 AA, and Number “ii” (−2) AA is a combination corresponding to a target DNA base or target DNA base sequence, and the combination of amino acids is determined according to any one of the following definitions:
(1-1) when Number 4 AA is glycine (G), Number 1 AA may be an arbitrary amino acid, and Number “ii” (−2) AA is aspartic acid (D), asparagine (N), or serine (S);
(1-2) when Number 4 AA is isoleucine (I), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid;
(1-3) when Number 4 AA is leucine (L), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid;
(1-4) when Number 4 AA is methionine (M), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid;
(1-5) when Number 4 AA is asparagine (N), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid;
(1-6) when Number 4 AA is proline (P), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid;
(1-7) when Number 4 AA is serine (S), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid;
(1-8) when Number 4 AA is threonine (T), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid; and
(1-9) when Number 4 AA is valine (V), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the combination of the three amino acids of Number 1 AA, Number 4 AA, and Number “ii” (−2) AA is a combination corresponding to a target DNA base or target DNA base sequence, and the combination of amino acids is determined according to any one of the following definitions:
(2-1) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA are an arbitrary amino acid, glycine, and aspartic acid, respectively, the PPR motif selectively binds to G;
(2-2) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are glutamic acid, glycine, and aspartic acid, respectively, the PPR motif selectively binds to G;
(2-3) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, glycine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-4) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are glutamic acid, glycine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-5) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, glycine, and serine, respectively, the PPR motif selectively binds to A, and next binds to C;
(2-6) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, isoleucine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T and C;
(2-7) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, isoleucine, and asparagine, respectively, the PPR motif selectively binds to T, and next binds to C;
(2-8) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, leucine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T and C;
(2-9) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, leucine, and aspartic acid, respectively, the PPR motif selectively binds to C;
(2-10) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, leucine, and lysine, respectively, the PPR motif selectively binds to T;
(2-11) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, methionine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T;
(2-12) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, methionine, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-13) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are isoleucine, methionine, and aspartic acid, respectively, the PPR motif selectively binds to T, and next binds to C;
(2-14) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, asparagine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to C and T;
(2-15) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-16) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are phenylalanine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-17) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are glycine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-18) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are isoleucine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-19) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are threonine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-20) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA are valine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T, and next binds to C;
(2-21) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA are tyrosine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T, and next binds to C;
(2-22) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, asparagine, and asparagine, respectively, the PPR motif selectively binds to C;
(2-23) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are isoleucine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C;
(2-24) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are serine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C;
(2-25) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are valine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C;
(2-26) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, asparagine, and serine, respectively, the PPR motif selectively binds to C;
(2-27) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are valine, asparagine, and serine, respectively, the PPR motif selectively binds to C;
(2-28) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, asparagine, and threonine, respectively, the PPR motif selectively binds to C;
(2-29) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are valine, asparagine, and threonine, respectively, the PPR motif selectively binds to C;
(2-30) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, asparagine, and tryptophan, respectively, the PPR motif selectively binds to C, and next binds to T;
(2-31) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are isoleucine, asparagine, and tryptophan, respectively, the PPR motif selectively binds to T, and next binds to C;
(2-32) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, proline, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T;
(2-33) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, proline, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-34) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are phenylalanine, proline, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-35) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are tyrosine, proline, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-36) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, serine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to A and G;
(2-37) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, serine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-38) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are phenylalanine, serine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-39) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are valine, serine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-40) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, threonine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to A and G;
(2-41) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, threonine, and aspartic acid, respectively, the PPR motif selectively binds to G;
(2-42) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are valine, threonine, and aspartic acid, respectively, the PPR motif selectively binds to G;
(2-43) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, threonine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-44) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are phenylalanine, threonine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-45) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are isoleucine, threonine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-46) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are valine, threonine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-47) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, valine, and an arbitrary amino acid, respectively, the PPR motif binds with A, C, and T, but does not bind to G;
(2-48) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are isoleucine, valine, and aspartic acid, respectively, the PPR motif selectively binds to C, and next binds to A;
(2-49) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, valine, and glycine, respectively, the PPR motif selectively binds to C; and
(2-50) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, valine, and threonine, respectively, the PPR motif selectively binds to T.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the one or more PPR motifs are any selected from
9 PPR motifs belonging to the p63 protein consisting of the amino acid sequence of SEQ ID NO: 1,
11 PPR motifs belonging to the GUN1 protein consisting of the amino acid sequence of SEQ ID NO: 2,
15 PPR motifs belonging to the pTac2 protein consisting of the amino acid sequence of SEQ ID NO: 3,
10 PPR motifs belonging to the DG1 protein consisting of the amino acid sequence of SEQ ID NO: 4, and
11 PPR motifs belonging to the GRP23 protein consisting of the amino acid sequence of SEQ ID NO: 5.

### Claim 5 (depends on 1)

A method for controlling a function of DNA, the method comprising:
preparing a DNA-binding protein by the method according to claim 1, and
allowing the protein to bind to a DNA molecule having a target DNA base or target DNA base sequence to control a function of the DNA molecule.

### Claim 6 (depends on 1)

A method for modifying a genetic substance of a cell, the method comprising:
preparing a DNA-binding protein by the method according to claim 1 as a fused protein comprising a functional region and a DNA binding region consisting of the DNA-binding protein;
preparing a cell containing a DNA having a target sequence; and
introducing the fused protein into the cell so that the DNA binding region of the fused protein binds to the DNA having a target sequence, and therefore the functional region modifies the DNA having a target sequence.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the functional region is fused to the DNA-binding protein on the C-terminus side of the protein.

### Claim 8 (depends on 6)

The method according to claim 6, wherein the functional region is a DNA-cleaving enzyme, or a nuclease domain thereof, or a transcription control domain, and the complex functions as a target sequence-specific DNA-cleaving enzyme or transcription control factor.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the DNA-cleaving enzyme is the nuclease domain of FokI (SEQ ID NO: 6).