METHODS FOR DESIGNING DNA BINDING PROTEIN CONTAINING PPR MOTIFS, AND USE THEREOF
20220064229 · 2022-03-03
Assignee
- KYUSHU UNIVERSITY, NAT'L UNIVERSITY CORPORATION (Fukuoka-shi, JP)
- Hiroshima University (Higashihiroshima-shi, Hiroshima, JP)
Inventors
- Takashi Yamamoto (Higashihiroshima-shi, JP)
- Tetsushi Sakuma (Higashihiroshima-shi, JP)
- Takahiro Nakamura (Fukuoka-shi, JP)
- Yusuke Yagi (Fukuoka-shi, JP)
- Yasuyuki Okawa (Fukuoka-shi, JP)
Cpc classification
C12N9/22
CHEMISTRY; METALLURGY
C07K2319/80
CHEMISTRY; METALLURGY
C12Y301/21004
CHEMISTRY; METALLURGY
C12N15/8213
CHEMISTRY; METALLURGY
International classification
C12N15/82
CHEMISTRY; METALLURGY
Abstract
A method for designing a protein capable of binding in a DNA base selective manner or DNA base sequence specific manner is provided. 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
1. A method for designing a DNA-binding protein that can bind in a DNA base-selective manner or a DNA base sequence-specific manner, the method comprising: determining an amino acid sequence of the DNA-binding protein, wherein the DNA-binding protein contains one or more 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 (M.sub.n+1) contiguously exists on the C-terminus side of the PPR motif (M.sub.n) (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 (M.sub.n); when a non-PPR motif consisting of 1 to 20 amino acids exists between the PPR motif (M.sub.n) and the next PPR motif (M.sub.n+1) on the C-terminus side, the amino acid locating upstream of the first amino acid of the next PPR motif (M.sub.n+1) by 2 positions, i.e., the −2nd amino acid; or when any next PPR motif (M.sub.n+1) does not exist on the C-terminus side of the PPR motif (M.sub.n), or 21 or more amino acids constituting a non-PPR motif exist between the PPR motif (M.sub.n) and the next PPR motif (M.sub.n+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 (M.sub.n) is referred to as Number “ii” (-2) amino acid (Number “ii” (-2) AA), each PPR motif (M.sub.n) contained in the protein is a PPR motif having a specific combination of amino acids as the three amino acids of Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, the combination of the three amino acids of Number 1 AA, Number 4 AA, and Number “ii” (-2) AA in each motif is a combination corresponding to a target DNA base of the target DNA base sequence, and the combination of amino acids is determined according to any one of the following definitions: (2-1) when the target DNA base to which the PPR motif binds is 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; (2-2) when the target DNA base to which the PPR motif binds is the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are glutamic acid, glycine, and aspartic acid, respectively; (2-3) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, glycine, and asparagine, respectively; (2-4) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are glutamic acid, glycine, and asparagine, respectively; (2-5) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, glycine, and serine, respectively; (2-6) when the target DNA base to which the PPR motif binds is T or C, 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; (2-7) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, isoleucine, and asparagine, respectively; (2-8) when the target DNA base to which the PPR motif binds is T or C, 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; (2-9) when the target DNA base to which the PPR motif binds is C, 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; (2-10) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, leucine, and lysine, respectively; (2-11) when the target DNA base to which the PPR motif binds is the target DNA base to which the PPR motif binds is T, 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; (2-12) when the target DNA base to which the PPR motif binds is T, 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; (2-13) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are isoleucine, methionine, and aspartic acid, respectively; (2-14) when the target DNA base to which the PPR motif binds is C or T, 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; (2-15) when the target DNA base to which the PPR motif binds is T, 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; (2-16) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are phenylalanine, asparagine, and aspartic acid, respectively; (2-17) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are glycine, asparagine, and aspartic acid, respectively; (2-18) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are isoleucine, asparagine, and aspartic acid, respectively; (2-19) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are threonine, asparagine, and aspartic acid, respectively; (2-20) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA are valine, asparagine, and aspartic acid, respectively; (2-21) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA are tyrosine, asparagine, and aspartic acid, respectively; (2-22) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, asparagine, and asparagine, respectively; (2-23) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are isoleucine, asparagine, and asparagine, respectively; (2-24) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are serine, asparagine, and asparagine, respectively; (2-25) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine, asparagine, and asparagine, respectively; (2-26) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, asparagine, and serine, respectively; (2-27) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine, asparagine, and serine, respectively; (2-28) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, asparagine, and threonine, respectively; (2-29) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine, asparagine, and threonine, respectively; (2-30) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, asparagine, and tryptophan, respectively; (2-31) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are isoleucine, asparagine, and tryptophan, respectively; (2-32) when the target DNA base to which the PPR motif binds is T, 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; (2-33) when the target DNA base to which the PPR motif binds is T, 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; (2-34) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are phenylalanine, proline, and aspartic acid, respectively; (2-35) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are tyrosine, proline, and aspartic acid, respectively; (2-36) when the target DNA base to which the PPR motif binds is A or 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; (2-37) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, serine, and asparagine, respectively; (2-38) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are phenylalanine, serine, and asparagine, respectively; (2-39) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine, serine, and asparagine, respectively; (2-40) when the target DNA base to which the PPR motif binds is A or 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; (2-41) when the target DNA base to which the PPR motif binds is 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; (2-42) when the target DNA base to which the PPR motif binds is the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine, threonine, and aspartic acid, respectively; (2-43) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, threonine, and asparagine, respectively; (2-44) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are phenylalanine, threonine, and asparagine, respectively; (2-45) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are isoleucine, threonine, and asparagine, respectively; (2-46) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine, threonine, and asparagine, respectively; (2-47) when the target DNA base to which the PPR motif binds is A, C, or T, 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; (2-48) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are isoleucine, valine, and aspartic acid, respectively; (2-49) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, valine, and glycine, respectively; and (2-50) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, valine, and threonine, respectively.
2. The method according to claim 1, wherein the one or more PPR motifs are any group of motifs 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.
3. A method for preparing the DNA-binding protein designed by the method according to claim 1, comprising: determining a nucleic acid sequence coding for an amino acid sequence of the designed DNA-binding protein, cloning said nucleic acid sequence, and preparing a transformant which produces the DNA-binding protein.
4. The method according to claim 3, wherein the one or more PPR motifs are any group of motifs 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.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0055]
[0056]
[0057]
[0058]
[0059]
[0060]
[0061]
[0062]
MODES FOR CARRYING OUT THE INVENTION
[0063] [PPR Motif and PPR Protein]
[0064] The “PPR motif” referred to in the present invention means a polypeptide constituted with 30 to 38 amino acids and having an amino acid sequence that shows, when the amino acid sequence is analyzed with a protein domain search program on the web (for example, Pfam, Prosite, Uniprot, etc.), an E value not larger than a predetermined value (desirably E-03) obtained at PF01535 in the case of Pfam (http://pfam.sanger.ac.uk/), or PS51375 in the case of Prosite (http://www.expasy.org/prosite/), unless otherwise indicated. The PPR motifs in various proteins are also defined in the Uniprot database (http://www.uniprot.org).
[0065] Although the amino acid sequence of the PPR motif is not highly conserved in the PPR motif of the present invention, such a secondary structure of helix, loop, helix, and loop as shown by the following formula is conserved well.
[Formula 2]
(Helix A)-X-(Helix B)-L (Formula 1)
[0066] The position numbers of the amino acids constituting the PPR motif defined in the present invention are according to those defined in a paper of the inventors of the present invention (Kobayashi K, et al., Nucleic Acids Res., 40, 2712-2723 (2012)). That is, the position numbers of the amino acids constituting the PPR motif defined in the present invention are substantially the same as the amino acid numbers defined for PF01535 in Pfam, but correspond to numbers obtained by subtracting 2 from the amino acid numbers defined for PS51375 in Prosite (for example, position 1 according to the present invention is position 3 of PS51375), and also correspond to numbers obtained by subtracting 2 from the amino acid numbers of the PPR motif defined in Uniprot.
[0067] More precisely, in the present invention, the No. 1 amino acid is the first amino acid from which Helix A shown in the formula 1 starts. The No. 4 amino acid is the fourth amino acid counted from the No. 1 amino acid. As for “ii” (-2)nd amino acid, [0068] when a next PPR motif (M.sub.n+1) contiguously exists on the C-terminus side of the PPR motif (M.sub.n) (when there is no amino acid insertion between the PPR motifs, as in the cases of, for example, Motif Nos. 1, 2, 3, 4, 6 and 7 in
[0071] The “PPR protein” referred to in the present invention means a PPR protein having two or more of the aforementioned PPR motifs, unless otherwise indicated. The term “protein” used in this specification means any substance consisting of a polypeptide (chain consisting of two or more amino acids bound through peptide bonds), and also includes those consisting of a comparatively low molecular weight polypeptide, unless otherwise indicated. The “amino acid” referred to in the present invention means a usual amino acid molecule, as well as an amino acid residue constituting a peptide chain. Which the term means will be apparent to those skilled in the art from the context.
[0072] Many PPR proteins exist in plants, and 500 proteins and about 5000 motifs can be found in Arabidopsis thaliana. PPR motifs and PPR proteins of various amino acid sequences also exist in many land plants such as rice, poplar, and selaginella. It is known that some PPR proteins are important factors for obtaining F1 seeds for hybrid vigor as fertility restoration factors that are involved in formation of pollen (male gamete). It has been clarified that some PPR proteins are involved in speciation, similarly in fertility restoration. It has also been clarified that almost all the PPR proteins act on RNA in mitochondria or chloroplasts.
[0073] It is known that, in animals, anomaly of the PPR protein identified as LRPPRC causes Leigh syndrome French Canadian (LSFC, Leigh's syndrome, subacute necrotizing encephalomyelopathy).
[0074] The term “selective” used for a property of a PPR motif for binding with a DNA base in the present invention means that a binding activity for any one base among the DNA bases is higher than binding activities for the other bases, unless otherwise indicates. Those skilled in the art can confirm this selectivity by planning an experiment, or it can also be obtained by calculation as described in the examples mentioned in this specification.
[0075] The DNA base referred to in the present invention means a base of deoxyribonucleotide constituting DNA, and specifically, it means any of adenine (A), guanine (G), cytosine (C), and thymine (T), unless otherwise indicated. Although the PPR protein may have selectivity to a base in DNA, it does not bind to a nucleic acid monomer.
[0076] Although search methods for conserved amino acid sequence as the PPR motif had been established before the present invention was accomplished, any rule concerning selective binding with DNA base had not been discovered at all.
[0077] [Findings Provided by the Present Invention]
[0078] The following findings are provided by the present invention.
(I) Information about Positions of Amino Acids Important for Selective Binding
[0079] Specifically, under the following definitions:
the first amino acid of Helix A of the PPR motif is referred to as No. 1 amino acid (No. 1 A.A.), the fourth amino acid as No. 4 amino acid (No. 4 A.A.), and [0080] when a next PPR motif (M.sub.n+1) contiguously exists on the C-terminus side of the PPR motif (M.sub.n) (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 (M.sub.n); [0081] when a non-PPR motif consisting of 1 to 20 amino acids exist between the PPR motif (M.sub.n) and the next PPR motif (M.sub.n+1) on the C-terminus side, the amino acid locating upstream of the first amino acid of the next PPR motif (M.sub.n+1) by 2 positions, i.e., the −2nd amino acid; or [0082] when any next PPR motif (M.sub.n+1) does not exist on the C-terminus side of the PPR motif (M.sub.n), or 21 or more amino acids constituting a non-PPR motif exist between the PPR motif (M.sub.n) and the next PPR motif (M.sub.n+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 (M.sub.n)
is referred to as No. “ii” (-2) amino acid (No. “ii” (-2) A.A.), combination of the three amino acids, the first and fourth amino acids of the helix (Helix A), No. 1 and No. 4 amino acids, and No. “ii” (-2) A.A. defined above (No. 1 A.A., No. 4 A.A. and No. “ii” (-2) A.A.) is important for selective binding to a DNA base, and to what kind of DNA base the motif binds can be determined on the basis of the combination.
[0083] The present invention is based on the findings concerning the combination of the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., found by the inventors of the present invention. Specifically:
(1-1) when No. 4 A.A. is glycine (G), No. 1 A.A. may be an arbitrary amino acid, No. “ii” (-2) A.A. is aspartic acid (D), asparagine (N), or serine (S), and the combination of No. 1 A.A., and No. “ii” (-2) A.A. may be, for example: [0084] a combination of an arbitrary amino acid and aspartic acid (D) (*GD), [0085] preferably a combination of glutamic acid (E) and aspartic acid (D) (EGD), [0086] a combination of an arbitrary amino acid and asparagine (N) (*GN), [0087] preferably a combination of glutamic acid (E) and asparagine (N) (EGN), or [0088] a combination of an arbitrary amino acid and serine (S) (*GS);
(1-2) when No. 4 A.A. is isoleucine (I), each of No. 1 A.A. and No. “ii” (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. “ii” (-2) A.A. may be, for example: [0089] a combination of an arbitrary amino acid and asparagine (N) (*IN);
(1-3) when No. 4 A.A. is leucine (L), each of No. 1 A.A. and No. “ii” (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. “ii” (-2) A.A. may be, for example: [0090] a combination of an arbitrary amino acid and aspartic acid (D) (*LD), or [0091] a combination of an arbitrary amino acid and lysine (K) (*LK);
(1-4) when No. 4 A.A. is methionine (M), each of No. 1 A.A. and No. “ii” (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. “ii” (-2) A.A. may be, for example: [0092] a combination of an arbitrary amino acid and aspartic acid (D) (*MD), or [0093] a combination of isoleucine (I) and aspartic acid (D) (IMD);
(1-5) when No. 4 A.A. is asparagine (N), each of No. 1 A.A. and No. “ii” (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. “ii” (-2) A.A. may be, for example: [0094] a combination of an arbitrary amino acid and aspartic acid (D) (*ND), [0095] a combination of any one of phenylalanine (F), glycine (G), isoleucine (I), threonine (T), valine
(V) and tyrosines (Y), and aspartic acid (D) (FND, GND, IND, TND, VND, or YND), [0096] a combination of an arbitrary amino acid and asparagine (N) (*NN), [0097] a combination of any one of isoleucine (I), serine (S) and valine (V), and asparagine (N) (INN, SNN or VNN) [0098] a combination of an arbitrary amino acid and serine (S) (*NS), [0099] a combination of valine (V) and serine (S) (VNS), [0100] a combination of an arbitrary amino acid and threonine (T) (*NT), [0101] a combination of valine (V) and threonine (T) (VNT), [0102] a combination of an arbitrary amino acid and tryptophan (W) (*NW), or [0103] a combination of isoleucine (I) and tryptophan (W) (INW);
(1-6) when No. 4 A.A. is proline (P), each of No. 1 A.A. and No. “ii” (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. “ii” (-2) A.A. may be, for example: [0104] a combination of an arbitrary amino acid and aspartic acid (D) (*PD), [0105] a combination of phenylalanine (F) and aspartic acid (D) (FPD), or [0106] a combination of tyrosine (Y) and aspartic acid (D) (YPD);
(1-7) when No. 4 A.A. is serine (S), each of No. 1 A.A. and No. “ii” (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. “ii” (-2) A.A. may be, for example: [0107] a combination of an arbitrary amino acid and asparagine (N) (*SN), [0108] a combination of phenylalanine (F) and asparagine (N) (FSN), or [0109] a combination of valine (V) and asparagine (N) (VSN);
(1-8) when No. 4 A.A. is threonine (T), each of No. 1 A.A. and No. “ii” (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. “ii” (-2) A.A. may be, for example: [0110] a combination of an arbitrary amino acid and aspartic acid (D) (*TD), [0111] a combination of valine (V) and aspartic acid (D) (VTD), [0112] a combination of an arbitrary amino acid and asparagine (N) (*TN), [0113] a combination of phenylalanine (F) and asparagine (N) (FTN), [0114] a combination of isoleucine (I) and asparagine (N) (ITN), or [0115] a combination of valine (V) and asparagine (N) (VTN); and
(1-9) when No. 4 A.A. is valine (V), each of No. 1 A.A. and No. “ii” (-2) A.A. may be an arbitrary amino acid, and the combination of No. 1 A.A., and No. “ii” (-2) A.A. may be, for example: [0116] a combination of isoleucine (I) and aspartic acid (D) (IVD), [0117] a combination of an arbitrary amino acid and glycine (G) (*VG), or [0118] a combination of an arbitrary amino acid and threonine (T) (*VT).
(II) Information about Correspondence of Combination of Three Amino Acids of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., and DNA Base
[0119] The protein is a protein determined on the basis of, specifically, the following definitions, and having a selective DNA base-binding property:
(2-1) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, glycine, and aspartic acid, respectively, the PPR motif selectively binds to G;
(2-2) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are glutamic acid, glycine, and aspartic acid, respectively, the PPR motif selectively binds to G;
(2-3) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, glycine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-4) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are glutamic acid, glycine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-5) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, leucine, and aspartic acid, respectively, the PPR motif selectively binds to C;
(2-10) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, leucine, and lysine, respectively, the PPR motif selectively binds to T;
(2-11) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, methionine, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-13) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-16) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are phenylalanine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-17) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are glycine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-18) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are isoleucine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-19) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are threonine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-20) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, asparagine, and asparagine, respectively, the PPR motif selectively binds to C;
(2-23) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are isoleucine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C;
(2-24) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are serine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C;
(2-25) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are valine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C;
(2-26) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, asparagine, and serine, respectively, the PPR motif selectively binds to C;
(2-27) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are valine, asparagine, and serine, respectively, the PPR motif selectively binds to C;
(2-28) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, asparagine, and threonine, respectively, the PPR motif selectively binds to C;
(2-29) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are valine, asparagine, and threonine, respectively, the PPR motif selectively binds to C;
(2-30) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, proline, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-34) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are phenylalanine, proline, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-35) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are tyrosine, proline, and aspartic acid, respectively, the PPR motif selectively binds to T;
(2-36) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, serine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-38) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are phenylalanine, serine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-39) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are valine, serine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-40) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, threonine, and aspartic acid, respectively, the PPR motif selectively binds to G;
(2-42) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are valine, threonine, and aspartic acid, respectively, the PPR motif selectively binds to G;
(2-43) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, threonine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-44) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are phenylalanine, threonine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-45) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are isoleucine, threonine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-46) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are valine, threonine, and asparagine, respectively, the PPR motif selectively binds to A;
(2-47) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., 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, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, valine, and glycine, respectively, the PPR motif selectively binds to C; and
(2-50) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., are an arbitrary amino acid, valine, and threonine, respectively, the PPR motif selectively binds to T.
[0120] Combination of amino acids of specific positions and binding property with a DNA base can be confirmed by experiments. Experiments for such purposes include preparation of a PPR motif or a protein containing two or more PPR motifs, preparation of a substrate DNA, and binding property test (for example, gel shift assay). These experiments are well known to those skilled in the art, and as for more specific procedures and conditions, for example, Patent document 2 can be referred to.
[0121] [Use of PPR Motif and PPR Protein]
[0122] Identification and Design
[0123] One PPR motif recognizes a specific one kind of base of DNA, and two or more contiguous PPR motifs can recognize continuous bases in a DNA sequence. Further, according to the present invention, by appropriately choosing amino acids at specific positions, PPR motifs selective for each of A, T, and C can be chosen or designed, and a protein containing an appropriate continuation of such PPR motifs can recognize a corresponding specific sequence. Therefore, according to the present invention, a naturally occurring PPR protein that selectively binds to DNA having a specific base sequence can be predicted or identified, or conversely, DNA as a target of binding of a PPR protein can be predicted and identified. Prediction or identification of such a target is useful for clarifying genetic identity of the target, and is also useful from a viewpoint that such prediction or identification may expand applicability of the target.
[0124] Furthermore, according to the present invention, a PPR motif that can selectively bind to a desired DNA base, and a protein having two or more PPR motifs that can bind to a desired DNA in a sequence-specific manner can be designed. In such design, as for the part other than the amino acids at the important positions in the PPR motif, sequence information on PPR motifs of naturally occurring type in DNA-binding type PPR proteins such as those of SEQ ID NOS: 1 to 5 can be referred to. Further, the motif or protein may also be designed by using a motif or protein of naturally occurring type as a whole, and replacing only the amino acids of the corresponding positions. Although the number of repetitions of PPR motifs can be appropriately chosen according to a target sequence, it may be, for example, 2 or more, preferably 2 to 30, more preferably 5 to 25, most preferably 9 to 15.
[0125] In the designing, amino acids other than those of the combination of the amino acids of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. may be taken into consideration. For example, selection of the amino acids of No. 8 and No. 12 described in Patent document 2 mentioned above may be important for exhibiting a DNA-binding activity. According to the researches of the inventors of the present invention, the No. 8 amino acid of a certain PPR motif and the No. 12 amino acid of the same PPR motif may cooperate in binding with DNA. The No. 8 amino acid may be a basic amino acid, preferably lysine, or an acidic amino acid, preferably aspartic acid, and the No. 12 amino acid may be a basic amino acid, neutral amino acid, or hydrophobic amino acid.
[0126] A designed motif or protein can be prepared by methods well known to those skilled in the art. That is, the present invention provides a PPR motif that selectively binds to a specific DNA base, and a PPR protein that specifically binds to DNA having a specific sequence, in which attention is paid to the combination of the amino acids of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. Such a motif and protein can be prepared even in a comparatively large amount by methods well known to those skilled in the art, and such methods may comprise determining a nucleic acid sequence encoding a target motif or protein from the amino acid sequence of the target motif or protein, cloning it, and preparing a transformant that produces the target motif or protein.
[0127] Preparation of Complex and Use Thereof
[0128] The PPR motif or PPR protein provided by the present invention can be made into a complex by binding a functional region. The functional region generally refers to a part having such a function as a specific biological function exerted in a living body or cell, for example, enzymatic function, catalytic function, inhibitory function, promotion function, etc, or a function as a marker. Such a region consists of, for example, a protein, peptide, nucleic acid, physiologically active substance, or drug.
[0129] According to the present invention, by binding a functional region to the PPR protein, the target DNA sequence-binding function exerted by the PPR protein, and the function exerted by the functional region can be exhibited in combination. For example, if a protein having a DNA-cleaving function (for example, restriction enzyme such as FokI) or a nuclease domain thereof is used as the functional region, the complex can function as an artificial DNA-cleaving enzyme.
[0130] In order to produce such a complex, methods generally available in this technical field can be used, and there are known a method of synthesizing such a complex as one protein molecule, a method of separately synthesizing two or more members of proteins, and then combining them to form a complex, and so forth.
[0131] In the case of the method of synthesizing a complex as one protein molecule, for example, a protein complex can be designed so as to comprise a PPR protein and a cleaving enzyme bound to the C-terminus of the PPR protein via an amino acid linker, an expression vector structure for expressing the protein complex can be constructed, and the target complex can be expressed from the structure. As such a preparation method, the method described in Japanese Patent Application No. 2011-242250, and so forth can be used.
[0132] For binding the PPR protein and the functional region protein, any binding means known in this technical field may be used, including binding via an amino acid linker, binding utilizing specific affinity such as binding between avidin and biotin, binding utilizing another chemical linker, and so forth.
[0133] The functional region usable in the present invention refers to a region that can impart any one of various functions such as those for cleavage, transcription, replication, restoration, synthesis, or modification of DNA, and so forth. By choosing the sequence of the PPR motif to define a DNA base sequence as a target, which is the characteristic of the present invention, substantially any DNA sequence may be used as the target, and with such a target, genome edition utilizing the function of the functional region such as those for cleavage, transcription, replication, restoration, synthesis, or modification of DNA can be realized.
[0134] For example, when the function of the functional region is a DNA cleavage function, there is provided a complex comprising a PPR protein part prepared according to the present invention and a DNA cleavage region bound together. Such a complex can function as an artificial DNA-cleaving enzyme that recognizes a base sequence of DNA as a target by the PPR protein part, and then cleaves DNA by the DNA cleavage region.
[0135] An example of the functional region having a cleavage function usable for the present invention is a deoxyribonuclease (DNase), which functions as an endodeoxyribonuclease. As such a DNase, for example, endodeoxyribonucleases such as DNase A (e.g., bovine pancreatic ribonuclease A, PDB 2AAS), DNase H and DNase I, restriction enzymes derived from various bacteria (for example, FokI (SEQ ID NO: 6) etc.) and nuclease domains thereof can be used. Such a complex comprising a PPR protein and a functional region does not exist in the nature, and is novel.
[0136] When the function of the functional region is a transcription control function, there is provided a complex comprising a PPR protein part prepared according to the present invention and a DNA transcription control region bound together. Such a complex can function as an artificial transcription control factor, which recognizes a base sequence of DNA as a target by the PPR protein part, and then controls transcription of the target DNA.
[0137] The functional region having a transcription control function usable for the present invention may be a domain that activates transcription, or may be a domain that suppresses transcription. Examples of the transcription control domain include VP16, VP64, TA2, STAT-6, and p65. Such a complex comprising a PPR protein and a transcription control domain does not exist in the nature, and is novel.
[0138] Further, the complex obtainable according to the present invention may deliver a functional region in a living body or cell in a DNA sequence-specific manner, and allow it to function. It thereby makes it possible to perform modification or disruption in a DNA sequence-specific manner in a living body or cell, like protein complexes utilizing a zinc finger protein (Non-patent documents 1 and 2 mentioned above) or TAL effecter (Non-patent document 3 and Patent document 1 mentioned above), and thus it becomes possible to impart a novel function, i.e., function for cleavage of DNA and genome edition utilizing that function. Specifically, with a PPR protein comprising two or more PPR motifs that can bind with a specific base linked together, a specific DNA sequence can be recognized. Then, genome edition of the recognized DNA region can be realized by the functional region bound to the PPR protein using the function of the functional region.
[0139] Furthermore, by binding a drug to the PPR protein that binds to a DNA sequence in a DNA sequence-specific manner, the drug may be delivered to the neighborhood of the DNA sequence as the target. Therefore, the present invention provides a method for DNA sequence-specific delivery of a functional substance.
[0140] It has been clarified that the PPR protein used as a material in the present invention works to specify an edition position for DNA edition, and such a PPR motif having specific amino acids arranged at the positions of the residues of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. recognizes a specific base on DNA, and then exhibits the DNA-binding activity thereof. On the basis of such a characteristic, a PPR protein of this type that has specific amino acids arranged at the positions of the residues of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. can be expected to recognize a base on DNA specific to each PPR protein, and as a result, introduce base polymorphism, or to be used in a treatment of a disease or condition resulting from a base polymorphism, and in addition, it is considered that the combination of such a PPR protein with such another functional region as mentioned above contribute to modification or improvement of functions for realizing cleavage of DNA for genome edition.
[0141] Moreover, an exogenous DNA-cleaving enzyme can be fused to the C-terminus of the PPR protein. Alternatively, by improving binding DNA base selectivity of the PPR motif on the N-terminus side, a DNA sequence-specific DNA-cleaving enzyme can also be constituted. Moreover, such a complex to which a marker part such as GFP is bound can also be used for visualization of a desired DNA in vivo.
EXAMPLES
Example 1: Collection of PPR Proteins and Target Sequences Thereof Used for DNA Edition
[0142] By referring to the information provided in the prior art references (Non-patent documents 11 to 15), structures and functions of the p63 protein (SEQ ID NO: 1), GUN1 protein (SEQ ID NO: 2), pTac2 protein (SEQ ID NO: 3), DG1 protein (SEQ ID NO: 4), and GRP23 protein (SEQ ID NO: 5) were analyzed.
[0143] To the PPR motif structures in such proteins, amino acid numbers defined in the present invention were imparted together with the information of the Uniprot database (http://www.uniprot.org/). The PPR motifs contained in the five kinds of PPR proteins of Arabidopsis thaliana (SEQ ID NOS: 1 to 5) used for the experiment, and the amino acid numbers thereof are shown in
[0144] Specifically, amino acid frequencies for the amino acids at the three positions (No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A.) responsible for the nucleic acid recognition codes in the PPR motifs considered to be important at the time of targeting RNA in the aforementioned p63 protein (SEQ ID NO: 1), GUN1 protein (SEQ ID NO: 2), pTac2 protein (SEQ ID NO: 3), DG1 protein (SEQ ID NO: 4), and GRP23 protein (SEQ ID NO: 5) were compared with those of RNA-binding type motifs.
[0145] The p63 protein of Arabidopsis thaliana (SEQ ID NO: 1) has 9 PPR motifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. in the amino acid sequence are as summarized in the following table and
TABLE-US-00001 TABLE 1 Code Base to be (1, 4, bound (ratio) A.sub.1 A.sub.4 L.sub.ii ii) A C G T PPR 230, V 233, R 263, S *R* 0.25 0.07 0.06 0.62 motif 1 PPR 265, F 368, D 297, S *D* 0.25 0.24 0.23 0.29 motif 2 PPR 299, L 302, K 332, D *KD 0.20 0.18 0.28 0.34 motif 3 PPR 334, Q 337, A 367, N *AN 0.45 0.18 0.05 0.32 motif 4 PPR 369, R 372, K 399, Y *K* 0.17 0.32 0.23 0.29 motif 5 PPR 401, E 404, L 434, S *LS 0.22 0.37 0.06 0.34 motif 6 PPR 436, S 439, S 469, E *SE 0.58 0.07 0.10 0.25 motif 7 PPR 471, T 474, D 505, M *D* 0.25 0.24 0.23 0.29 motif 8 PPR 507, N 510, M 540, R *M* 0.13 0.14 0.22 0.51 motif 9
[0146] The GUN1 protein of Arabidopsis thaliana (SEQ ID NO: 2) has 11 PPR motifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. in the amino acid sequence are as summarized in the following table and
TABLE-US-00002 TABLE 2 Code Base to be (1, 4, bound (ratio) A.sub.1 A.sub.4 L.sub.ii ii) A C G T PPR 234, K 237, S 267, 7 *S* 0.41 0.12 0.22 0.25 motif 1 PPR 269,Y 272, S 302, N *SN 0.62 0.07 0.04 0.26 motif 2 PPR 304, V 307, N 338, D VND 0.06 0.21 0.24 0.31 motif 3 PPR 340, I 343, N 373, D IND 0.14 0.24 0.12 0.50 motif 4 PPR 375, F 378, N 408, N FNN 0.24 0.21 0.24 0.31 motif 5 PPR 410, V 413, S 443, D VSD 0.33 0.24 0.23 0.20 motif 6 PPR 445, V 448, N 478, D VND 0.06 0.21 0.06 0.66 motif 7 PPR 480, V 483, N 513, N VNN 0.17 0.48 0.09 0.26 motif 8 PPR 515, L 518, S 548, D *SD 0.20 0.17 0.39 0.24 motif 9 PPR 550, V 553, S 583, N VSN 0.57 0.09 0.05 0.30 motif 10 PPR 585, V 588, N 620, A *N* 0.10 0.33 0.10 0.48 motif 11
[0147] The pTac2 protein of Arabidopsis thaliana (SEQ ID NO: 3) has 15 PPR motifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. in the amino acid sequence are as summarized in the following table and
TABLE-US-00003 TABLE 3 Code Base to be bound A.sub.1 A.sub.4 L.sub.ii (1, 4, ii) A C G T PPR 106, N 109, A 140, N *AN 0.45 0.18 0.05 0.32 motif 1 PPR 142, H 145, T 175, S *TS 0.37 0.29 0.15 0.19 motif 2 PPR 177, F 180, T 210, S *TS 0.37 0.29 0.15 0.19 motif 3 PPR 212, L 215, N 246, D LND 0.08 0.15 0.23 0.54 motif 4 PPR 248, V 251, N 281, D VND 0.06 0.21 0.06 0.66 motif 5 PPR 283, T 286, S 316, D TSD 0.14 0.18 0.14 0.54 motif 6 PPR 318, T 321, N 351, N TNN 0.08 0.49 0.17 0.26 motif 7 PPR 353, N 356, S 386, D *SD 0.20 0.17 0.39 0.24 motif 8 PPR 388, A 491, N 421, D AND 0.07 0.05 0.14 0.74 motif 9 PPR 423, E 426, E 456, S B.G. 0.25 0.21 0.18 0.36 motif 10 PPR 458, K 461, T 491, S *TS 0.37 0.29 0.15 0.19 motif 11 PPR 493, E 496, H 526, N *H* 0.17 0.34 0.06 0.43 motif 12 PPR 528, D 531, N 561, D *ND 0.11 0.17 0.10 0.62 motif 13 PPR 563, R 566, E 596, S B.G. 0.25 0.21 0.18 0.36 motif 14 PPR 598, M 601, C 631, I *C* 0.55 0.10 0.21 0.14 motif 15
[0148] The DG1 protein of Arabidopsis thaliana (SEQ ID NO: 4) has 10 PPR motifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. in the amino acid sequence are as summarized in the following table and
TABLE-US-00004 TABLE 4 Code Base to be bound A.sub.1 A.sub.4 L.sub.ii (1, 4, ii) A C G T PPR 256, F 259, T 290, D *TD 0.10 0.10 0.67 0.13 motif 1 PPR 292, A 295, H 340, D *H* 0.17 0.34 0.06 0.43 motif 2 PPR 342, V 345, N 375, N VNN 0.17 0.48 0.09 0.26 motif 3 PPR 377, A 380, G 410, K *G* 0.29 0.13 0.31 0.27 motif 4 PPR 412, I 415, K 445, T *K* 0.17 0.32 0.23 0.29 motif 5 PPR 447, S 450, Y 481, L B.G. 0.25 0.21 0.18 0.36 motif 6 PPR 483, I 486, T 515, N ITN 0.79 0.06 0.05 0.10 motif 7 PPR 517, G 520, N 553, N *NN 0.12 0.44 0.13 0.30 motif 8 PPR 555, Y 558, S 588, D YSD 0.25 0.15 0.39 0.20 motif 9 PPR 590, T 593, A 623, H *AH 0.41 0.08 0.07 0.45 motif 10
[0149] The GRP23 protein of Arabidopsis thaliana (SEQ ID NO: 5) has 11 PPR motifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. in the amino acid sequence are as summarized in the following table and
TABLE-US-00005 TABLE 5 Code Base to be bound A.sub.1 A.sub.4 L.sub.ii (1, 4, ii) A C G T PPR 181, F 184, N 215, N FNN 0.24 0.21 0.24 0.31 motif 1 PPR 217, V 220, N 251, S VNS 0.07 0.61 0.05 0.27 motif 2 PPR 253, V 256, R 286, D *RD 0.25 0.07 0.06 0.62 motif 3 PPR 288, T 291, N 321, D TND 0.14 0.08 0.07 0.71 motif 4 PPR 323, I 326, A 356, H *AH 0.41 0.08 0.07 0.45 motif 5 PPR 358, P 361, N 396, N *NN 0.12 0.44 0.13 0.30 motif 6 PPR 398, D 401, G 435, D *GD 0.09 0.09 0.59 0.25 motif 7 PPR 437, L 440, C 470, D *CD 0.30 0.15 0.35 0.20 motif 8 PPR 472, P 475, R 505, V *R* 0.25 0.07 0.06 0.62 motif 9 PPR 507, D 510, A 540, D *AD 0.10 0.22 0.39 0.29 motif 10 PPR 542, S 545, D 575, T *D* 0.25 0.24 0.23 0.29 motif 11
[0150] The amino acid frequencies for these positions were confirmed for each protein, and compared with the amino acid frequencies for the same positions of the RNA-binding type motifs. The results are shown in
[0151] With reference to the RNA recognition codes described in the non-patent document (Yagi, Y. et al., Plos One, 2013, 8, e57286), the DNA-binding type PPR motifs that selectively bind to each corresponding base were evaluated. More precisely, a chi square test was performed on the basis of occurrence nucleotide frequencies shown in Table 6 and expected nucleotide frequencies calculated from the background frequencies. The test was performed for each base (NT), purine or pyrimidine (AG or CT, PY), hydrogen bond group (AT or GC, HB), or amino or keto form (AC or GT). Significant value was defined as P<0.06 (5E-02, 5% significance level), and when a significant value was obtained in any of the tests, the combination of No. 1 amino acid, No. 4 amino acid, and No. “ii” (-2) amino acid was chosen.
TABLE-US-00006 TABLE 6 Base selectivity of DNA-binding code NSRs occurrence Probabilitiy matrix subtraction for background (1, 4, ii) of the NSR(s) A C G T A C G T *GD 14 0.10 0.06 0.57 0.28 −0.16 −0.15 0.40 −0.08 EGD 8 0.07 0.05 0.69 0.19 −1.19 −1.16 0.52 −0.17 *GN 11 0.55 0.10 0.04 0.31 0.29 −0.11 −0.13 −0.05 EGN 5 0.63 0.06 0.05 0.25 0.37 −0.15 −0.12 −0.11 *GS 3 0.57 0.23 0.06 0.14 0.31 0.02 −0.11 −0.22 *I* 15 0.15 0.29 0.10 0.45 −0.11 0.08 −0.07 0.09 *IN 4 0.17 0.28 0.06 0.50 −0.09 0.07 −0.11 0.14 *L* 23 0.20 0.30 0.03 0.47 −0.06 0.09 −0.14 0.11 *LD 6 0.19 0.47 0.05 0.28 −0.07 0.26 −0.12 −0.08 *LK 3 0.09 0.08 0.06 0.77 −0.17 −0.13 −0.11 0.41 *M* 10 0.14 0.15 0.16 0.56 −0.12 −0.06 −0.02 0.20 *MD 9 0.15 0.13 0.17 0.55 −0.11 −0.08 0.00 0.19 IMD 4 0.09 0.24 0.06 0.62 −0.17 0.03 −0.11 0.26 *N* 147 0.11 0.33 0.10 0.45 −0.15 0.12 −0.07 0.09 ND 72 0.11 0.18 0.10 0.61 −0.15 −0.03 −0.07 0.25 FND 13 0.23 0.19 0.10 0.49 −0.03 −0.02 −0.07 0.13 GND 3 0.09 0.08 0.06 0.77 −0.17 −0.13 −0.11 0.41 IND 5 0.22 0.13 0.06 0.60 −0.04 −0.08 −0.12 0.24 TND 3 0.15 0.08 0.06 0.72 −0.11 −0.13 −0.11 0.36 VND 23 0.06 0.25 0.06 0.63 −0.20 0.04 −0.11 0.27 YND 6 0.08 0.30 0.11 0.52 −0.18 0.09 −0.06 0.16 *NN 34 0.15 0.45 0.14 0.27 −0.11 0.24 −0.03 −0.09 INN 7 0.12 0.49 0.05 0.34 −0.14 0.28 −0.12 −0.02 SNN 3 0.09 0.60 0.06 0.24 −0.17 0.39 −0.11 −0.12 VNN 10 0.20 0.53 0.04 0.23 −0.06 0.32 −0.13 −0.13 *NS 13 0.11 0.47 0.07 0.36 −0.15 0.26 −0.10 0.00 VNS 5 0.08 0.66 0.05 0.21 −0.18 0.45 −0.12 −0.15 *NT 13 0.12 0.52 0.13 0.24 −0.14 0.31 −0.04 −0.12 VNT 5 0.08 0.57 0.05 0.30 −0.18 0.36 −0.12 −0.06 *NW 11 0.14 0.32 0.13 0.41 −0.12 0.11 −0.04 0.05 INW 3 0.09 0.29 0.06 0.56 −0.17 0.08 −0.11 0.20 *P* 17 0.10 0.06 0.11 0.73 −0.16 −0.15 −0.06 0.37 *PD 9 0.06 0.09 0.10 0.75 −0.20 −0.12 −0.07 0.39 FPD 3 0.09 0.08 0.06 0.77 −0.17 −0.13 −0.11 0.41 YPD 3 0.09 0.08 0.06 0.77 −0.17 −0.13 −0.11 0.41 *S* 49 0.38 0.13 0.20 0.29 0.12 −0.08 0.03 −0.07 *SN 18 0.63 0.08 0.05 0.24 0.37 −0.13 −0.12 −0.12 FSN 7 0.63 0.13 0.08 0.16 0.37 −0.08 −0.09 −0.20 VSN 6 0.60 0.10 0.05 0.25 0.34 −0.11 −0.12 −0.11 *T* 86 0.45 0.09 0.31 0.15 0.19 −0.12 0.14 −0.21 *TD 32 0.13 0.12 0.61 0.14 −0.13 −0.09 0.44 −0.22 VTD 7 0.07 0.06 0.67 0.20 −0.19 −0.15 0.50 −0.16 *TN 31 0.66 0.08 0.13 0.13 0.40 −0.13 −0.04 −0.23 FTN 4 0.75 0.07 0.06 0.12 0.49 −0.14 −0.11 −0.24 ITN 5 0.77 0.06 0.05 0.11 0.51 −0.15 −0.12 −0.25 VTN 10 0.63 0.13 0.15 0.09 0.37 −0.08 −0.02 −0.27 *V* 48 0.29 0.21 0.08 0.43 0.03 0.00 −0.09 0.07 IVD 3 0.31 0.50 0.06 0.14 0.05 0.29 −0.11 −0.22 VG 5 0.22 0.48 0.05 0.25 −0.04 0.27 −0.12 −0.11 *VT 4 0.25 0.07 0.06 0.62 −0.01 −0.14 −0.11 0.26 Background frequency 0.26 0.21 0.17 0.36
[0152] In Table 1, the combinations of the amino acids that showed significant base selectivity were mentioned. That is, these results mean that the PPR motifs having the amino acid species of the No. 1 amino acid, No. 4 amino acid, and No. “ii” (-2) amino acid (“NSRs (1, 4, and ii)” in the table) that provided a significant P value are PPR motifs that impart base-selective binding ability, and a larger “positive” value obtained after the subtraction of the background means higher base selectivity for the base. Among the No. 1 amino acid, No. 4 amino acid, and No. “ii” (-2) amino acid, the No. 4 amino acid most strongly affects the base selectivity, the No. “ii” (-2) amino acid affects the base selectivity next strongly, and the No. 1 amino acid most weakly affects the base selectivity among the three amino acids.
Example 2: Evaluation of Sequence-Specific DNA-Binding Ability PPR Molecules
[0153] In this example, artificial transcription factors were prepared by fusing VP64, which is a transcription activation domain, to the three kinds of DNA-binding type (expectedly) PPR molecules, p63, pTac2, and GUN1, and by examining whether they could activate luciferase reporters each having a corresponding target sequence in a human cultured cell, whether the PPR molecules had a sequence-specific DNA-binding ability or not was determined (
[0154] (Experimental Method)
[0155] 1. Preparation of PPR-VP64 Expression Vector
[0156] Only the parts corresponding to the PPR motifs in the coding sequences of p63, pTac2, and GUN1 were prepared by artificial synthesis. For the DNA synthesis, the artificial gene synthesis service of Biomatik was used. The pCS2P vector having the CMV promoter was used as a backbone vector, and each synthesized PPR sequence was inserted into it. Further, the Flag tag and nuclear transfer signal were inserted at the N-terminus of the PPR sequence, and the VP64 sequence was inserted at the C-terminus of the same. The produced sequences of p63-VP64, pTac2-VP64, and GUN1-VP64 are shown in Sequence Listing as SEQ ID NOS: 7 to 9.
[0157] 2. Preparation of Reporter Vector Having PPR Target Sequence
[0158] A reporter vector (pminCMV-luc2, SEQ ID NO: 10) was prepared, in which the firefly luciferase gene was ligated downstream from the Minimal CMV promoter, and a multi-cloning site was placed upstream of the promoter. The predicted target sequence of each PPR was inserted into the vector at the multi-cloning site. The target sequence of each PPR (TCTATCACT for p63, AACTTTCGTCACTCA for pTac2, and AATTTGTCGAT for GUN1, SEQ ID NOS: 11 to 13 in Sequence Listing) was determined by predicting the motif-DNA recognition codes of DNA-binding type PPR from the motif-RNA recognition codes observed in the RNA-binding type PPR. For each PPR, sequences containing 4 or 8 of target sequences were prepared, and used in the following assay. The nucleotide sequences of the vectors are shown as SEQ ID NOS: 14 to 19 in Sequence Listing.
[0159] 3. Transfection into HEK293 T Cell
[0160] The PPR-VP64 expression vector prepared in the section 1, the firefly luciferase expression vector prepared in the section 2, and the pRL-CMV vector (expression vector for Renilla luciferase, Promega) as a reference were introduced by using Lipofectamine LTX (Life Technologies). The DMEM medium (25 μl) was added to each well of a 96-well plate, and a mixture containing the PPR-VP64 expression vector (400 ng), firefly luciferase expression vector (100 ng), and pRL-CMV vector (20 ng) was further added. Then, a mixture of the DMEM medium (25 μl) and Lipofectamine LTX (0.7 μl) was added to each well, the plate was left standing at room temperature for 30 minutes, then 6×10.sup.4 of the HEK293 T cells suspended in the DMEM medium containing 15% fetal bovine serum (100 μl) were added, and the cells were cultured at 37° C. in a CO.sub.2 incubator for 24 hours.
[0161] 4. Luciferase Assay
[0162] Luciferase assay was performed by using Dual-Glo Luciferase Assay System (Promega) in accordance with the instructions attached to the kit. For the measurement of the luciferase activity, Tri Star LB 941 Plate Reader (Berthold) was used.
[0163] (Results and Discussion)
[0164] The luciferase activity was compared for the cases of introducing pTac2-VP64 or GUN1-VP64 together with pminCMV-luc2 for a negative control, or the reporter vector having 4 or 8 target sequences (table mentioned below,
TABLE-US-00007 Fluc reporter PPR-VP64 Reference Fluc Rluc Fluc/Rluc Fold activation pTac2-VP64 pminCMV-luc2 pTac2-VP64 pRL-CMV 47744 4948 9.649151172 1 (negative control) pTac2-VP64 pTac2-4x target pTac2-VP64 pRL-CMV 133465 4757 28.05654824 2.907670089 (4x target) pTac2-VP64 pTac2-8x target pTac2-VP64 pRL-CMV 189146 4011 47.15681875 4.887146849 (8x target) GUN1-VP64 pminCMV-luc2 GUN1-VP64 pRL-CMV 29590 3799 7.788891814 1 (negative control) GUN1-VP64 GUN1-4x target GUN1-VP64 pRL-CMV 61070 2727 22.39457279 2.875193715 (4x target) GUN1-VP64 GUN1-8x target GUN1-VP64 pRL-CMV 66982 2731 24.52654705 3.14891356 (8x target)