PPR proteins with low aggregation and their applications

Abstract

To provide a composition for treating diseases, comprising a vector containing a nucleic acid encoding a PPR protein with improved aggregation properties.SOLUTION: To improve the aggregation properties of PPR proteins, the A6 amino acid of the first PPR motif (M1) from the N-terminus is made more hydrophilic. Furthermore, the A9 amino acid of M1 is made a hydrophilic amino acid or glycine. The A6 amino acid is preferably asparagine or aspartic acid, and the A9 amino acid is preferably glutamine, glutamic acid, lysine, or glycine. Proteins containing such PPR motifs as the M1 motif not only have improved aggregation properties, but can also have high binding avidity for target nucleic acids.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a nucleic acid manipulation technique using a protein capable of binding to a desired nucleic acid. The present invention is useful in a wide range of fields such as medicine (drug discovery support, disease treatment), agriculture (agricultural product production, breeding), and chemistry (biological substance production).

Background Art

[0002] PPR proteins are proteins containing repeats of PPR motifs consisting of about 35 amino acids in length, and one PPR motif can specifically bind to one base. The combination of the first, fourth, and iith (two before the next motif) amino acids in the PPR motif determines which of adenine, cytosine, guanine, and uracil (or thymine) it binds to (Patent Documents 1 and 2).

[0003] Among the naturally occurring RNA-binding PPR motifs, the most frequently occurring combinations corresponding to each base are: for adenine, the first is valine, the fourth is threonine, and the iith is asparagine; for cytosine, the first is valine, the fourth is asparagine, and the iith is serine; for guanine, the first is valine, the fourth is threonine, and the iith is aspartic acid; for uracil, the first is valine, the fourth is asparagine, and the iith is aspartic acid (Non-Patent Documents 1 to 5). By using these amino acid combinations, it is possible to design PPR proteins that can specifically bind to any sequence.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0005] [Non-Patent Document 1] Coquille, S. et al. An artificial PPR scaffold for programmable RNA recognition. Nature Communications 5, Article number: 5729(2014) [Non-Patent Document 2] Shen, C. et al. Specific RNA Recognition by Designer Pentatricopeptide Repeat Protein. Molecular Plant 8, 667-670(2015) [Non-Patent Document 3] Shen, C. et al. Structural basis for specific single-stranded RNA recognition by designer pentatricopeptide repeat proteins. Nature Communications volume 7, Article number: 11285 (2016) [Non-Patent Document 4] Miranda, RG et al. RNA-binding specificity landscapes of designer pentatricopeptide repeat proteins elucidate principles of PPR-RNA interactions. Nucleic Acids Research, 46(5), 2613-2623(2018) [Non-Patent Document 5] Yan, J. et al. Delineation of pentatricopeptide repeat codes for target RNA prediction. Nucleic Acids Research, gkz075(2019) [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The inventors have been investigating how to produce high-performance PPR proteins with many linked PPR motifs, such as 15 or more, using the above-mentioned amino acid combinations (Patent Document 3). On the other hand, the inventors' investigations have shown that some PPR proteins produced by this method exhibit aggregation. In particular, aggregation was sometimes observed when PPR proteins were expressed in animal cultured cells. [Means for solving the problem]

[0007] Therefore, we considered resolving this issue through amino acid mutations within the PPR motif. We found that the aggregation properties of PPR can be improved by making the 6th, preferably the 6th and 9th, amino acids of the first motif (N-terminal side) of the PPR protein hydrophilic amino acids, thus completing the present invention.

[0008] The present invention provides the following: [1] One of the following PPR motifs: (C-1) Sequence ID: A PPR motif consisting of one of sequences 4-7; (C-2) Sequence ID: A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 are substituted, deleted, or added in any of the sequences of 4 to 7, and which is cytosine-binding; (C-3) Sequence ID: A PPR motif having at least 80% sequence identity with any one of sequences 4-7, wherein the amino acids at positions 1, 4, 6, and 34 are identical, and which is cytosine-binding; (A-1) A PPR motif consisting of sequence number 8, in which the amino acid at position 6 is substituted with asparagine or aspartic acid; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 in the sequence (A-2)(A-1) are substituted, deleted, or added, and which is adenine-binding; A PPR motif having at least 80% sequence identity with (A-3)(A-1), except that the amino acids at positions 1, 4, 6, and 34 are identical, and which is adenine-binding; (G-1) A PPR motif consisting of sequence number 9, in which the amino acid at position 6 is substituted with asparagine or aspartic acid; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 in the (G-2)(G-1) sequence are substituted, deleted, or added, and which is guanine-binding; A PPR motif having at least 80% sequence identity with (G-3)(G-1), except that the amino acids at positions 1, 4, 6, and 34 are identical, and which is guanine-binding; (U-1) A PPR motif consisting of sequence number 10, in which the amino acid at position 6 is substituted with asparagine or aspartic acid; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 in the (U-2)(U-1) sequence are substituted, deleted, or added, and which is uracil-binding; A PPR motif having at least 80% sequence identity with (U-3)(U-1), except that the amino acids at positions 1, 4, 6, and 34 are identical, and which is uracil-binding. [2] One of the following PPR motifs: (C-1) Sequence ID: A PPR motif consisting of one of sequences 4-7; (C-2) Sequence ID: A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 are substituted, deleted, or added in any of the sequences of 4 to 7, and which is cytosine-binding; (C-3) Sequence ID: A PPR motif having at least 80% sequence identity with any one of sequences 4-7, wherein the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is cytosine-binding; (A-1) A PPR motif obtained by substituting the amino acids at positions 6 and 9 in the sequence of sequence number 8 in such a way that one of the combinations defined below is satisfied; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 in the (A-2)(A-1) sequence are substituted, deleted, or added, and which is adenine-binding; A PPR motif having at least 80% sequence identity with (A-3)(A-1), except that the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is adenine-binding; (G-1) A PPR motif consisting of a sequence in which the amino acids at positions 6 and 9 in sequence number 9 are substituted to satisfy one of the combinations defined below; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 in the (G-2)(G-1) sequence are substituted, deleted, or added, and which is guanine-binding; A PPR motif having at least 80% sequence identity with (G-3)(G-1), except that the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is guanine-binding; (U-1) A PPR motif consisting of a sequence in which the amino acids at positions 6 and 9 in sequence number 10 are substituted to satisfy one of the combinations defined below; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 in the (U-2)(U-1) sequence are substituted, deleted, or added, and which is uracil-binding; A PPR motif having at least 80% sequence identity with (U-3)(U-1), except that the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is uracil-binding. • A combination in which the amino acid at position 6 is asparagine and the amino acid at position 9 is glutamic acid. • A combination where the amino acid at position 6 is asparagine and the amino acid at position 9 is glutamine. • A combination in which the amino acid at position 6 is asparagine and the amino acid at position 9 is lysine. • A combination where the amino acid at position 6 is aspartic acid, and the amino acid at position 9 is glycine. [3] One of the following PPR motifs described in 1 or 2: (C-4) Sequence number: A PPR motif consisting of a sequence of 4; (A-4) Sequence ID: PPR motif consisting of sequence 58; (G-4) Sequence ID: PPR motif consisting of sequence 59; (U-4) Sequence number: A PPR motif consisting of sequence 60. [4] Use of any one of the PPR motifs described in items 1-3 as the first PPR motif from the N-terminus in a PPR protein. [5] Use as described in 4 to reduce the aggregation of PPR proteins. [6] A protein capable of binding to a target nucleic acid having a specific base sequence, comprising 1 to 30 PPR motifs represented by formula 1 below, wherein the A6 amino acid of the first PPR motif from the N-terminus (M1) is a hydrophilic amino acid. [ka] (In the formula: Helix A is a 12-amino acid length region capable of forming an α-helix structure, represented by Equation 2. [ka] In formula 2, A1~A 12 Each of these independently represents an amino acid; X is either absent or a portion consisting of 1 to 9 amino acids in length; Helix B is a region consisting of 11-13 amino acids that can form an α-helix structure; L is the portion represented by formula 3, which is 2 to 7 amino acids long; [ka] In Equation 3, each amino acid is numbered from the C-terminus as "i" (-1), "ii" (-2), and so on. However, L iii ~L vii (It may not exist.) [7] The protein according to 6, wherein the A9 amino acid of M1 is a hydrophilic amino acid or glycine. [8] The protein according to 6 or 7, wherein the A6 amino acid of M1 is asparagine or aspartic acid. [9] A protein according to any one of items 6-8, wherein the A9 amino acid of M1 is glutamine, glutamic acid, lysine, or glycine.

[10] A protein described in any one of items 6-9, wherein the A6 amino acid of M1 and the A9 amino acid of M1 are in any of the following combinations. • The combination in which amino acid A6 is asparagine and amino acid A9 is glutamic acid. • The combination in which amino acid A6 is asparagine and amino acid A9 is glutamine. • A combination where amino acid A6 is asparagine and amino acid A9 is lysine. • A combination where amino acid A6 is aspartic acid and amino acid A9 is glycine.

[11] A fusion protein comprising at least one selected from the group consisting of a fluorescent protein, a nuclear localization signal peptide, and a tag protein, and a PPR protein containing the PPR motif described in any one of items 1 to 3 as the first PPR motif from the N-terminus, or a protein described in any one of items 6 to 10.

[12] A method for modifying a PPR protein that can bind to a target nucleic acid having a specific base sequence, including the PPR motif defined in 6, wherein the A6 amino acid of the first PPR motif from the N-terminus (M1) is made more hydrophilic.

[13] A method for detecting nucleic acids, characterized by using a PPR protein containing one of the PPR motifs described in any one of items 1 to 3 as the first PPR motif from the N-terminus, a protein described in any one of items 6 to 10, or a fusion protein described in item 11.

[14] A nucleic acid encoding a PPR motif as described in any one of items 1 to 3, a PPR protein containing the PPR motif described in any one of items 1 to 3 as the first PPR motif from the N-terminus, or a protein as described in any one of items 6 to 10.

[15] A vector containing the nucleic acid described in 14.

[16] Cells (excluding human individuals) containing the vector described in 15.

[17] A method for manipulating nucleic acids (excluding implementation in human organisms) using any one of the PPR motifs described in items 1 to 3, a PPR protein containing any one of the PPR motifs described in items 1 to 3 as the first PPR motif from the N-terminus, a protein described in any one of items 6 to 10, or a vector described in item 15.

[18] A method for producing an organism, including the operation described in 17.

[0009] The present invention also provides the following: [1] One of the following PPR motifs: (C-1) Sequence ID: A PPR motif consisting of one of sequences 4-7; (C-2) Sequence ID: A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 are substituted, deleted, or added in any of the sequences of 4 to 7, and which is cytosine-binding; (C-3) Sequence ID: A PPR motif having at least 80% sequence identity with any one of sequences 4-7, wherein the amino acids at positions 1, 4, 6, and 34 are identical, and which is cytosine-binding; (A-1) A PPR motif consisting of sequence number 8, in which the amino acid at position 6 is substituted with asparagine or aspartic acid; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 in the sequence (A-2)(A-1) are substituted, deleted, or added, and which is adenine-binding; A PPR motif having at least 80% sequence identity with (A-3)(A-1), except that the amino acids at positions 1, 4, 6, and 34 are identical, and which is adenine-binding; (G-1) A PPR motif consisting of sequence number 9, in which the amino acid at position 6 is substituted with asparagine or aspartic acid; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 in the (G-2)(G-1) sequence are substituted, deleted, or added, and which is guanine-binding; A PPR motif having at least 80% sequence identity with (G-3)(G-1), except that the amino acids at positions 1, 4, 6, and 34 are identical, and which is guanine-binding; (U-1) A PPR motif consisting of sequence number 10, in which the amino acid at position 6 is substituted with asparagine or aspartic acid; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 in the (U-2)(U-1) sequence are substituted, deleted, or added, and which is uracil-binding; A PPR motif having at least 80% sequence identity with (U-3)(U-1), except that the amino acids at positions 1, 4, 6, and 34 are identical, and which is uracil-binding. [2] One of the following PPR motifs: (C-1) Sequence ID: A PPR motif consisting of one of sequences 4-7; (C-2) Sequence ID: A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 are substituted, deleted, or added in any of the sequences of 4 to 7, and which is cytosine-binding; (C-3) Sequence ID: A PPR motif having at least 80% sequence identity with any one of sequences 4-7, wherein the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is cytosine-binding; (A-1) A PPR motif obtained by substituting the amino acids at positions 6 and 9 in the sequence of sequence number 8 in such a way that one of the combinations defined below is satisfied; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 in the (A-2)(A-1) sequence are substituted, deleted, or added, and which is adenine-binding; A PPR motif having at least 80% sequence identity with (A-3)(A-1), except that the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is adenine-binding; (G-1) A PPR motif consisting of a sequence in which the amino acids at positions 6 and 9 in sequence number 9 are substituted to satisfy one of the combinations defined below; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 in the (G-2)(G-1) sequence are substituted, deleted, or added, and which is guanine-binding; A PPR motif having at least 80% sequence identity with (G-3)(G-1), except that the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is guanine-binding; (U-1) A PPR motif consisting of a sequence in which the amino acids at positions 6 and 9 in sequence number 10 are substituted to satisfy one of the combinations defined below; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 in the (U-2)(U-1) sequence are substituted, deleted, or added, and which is uracil-binding; A PPR motif having at least 80% sequence identity with (U-3)(U-1), except that the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is uracil-binding. • A combination in which the amino acid at position 6 is asparagine and the amino acid at position 9 is glutamic acid. • A combination in which the amino acid at position 6 is asparagine and the amino acid at position 9 is glutamine. • A combination in which the amino acid at position 6 is asparagine and the amino acid at position 9 is lysine. • A combination in which the amino acid at position 6 is aspartic acid and the amino acid at position 9 is glycine. [3] Use of the PPR motif described in 1 or 2 as the first PPR motif from the N-terminus in a PPR protein. [4] Use as described in 3 to reduce the aggregation of PPR proteins. [5] A protein capable of binding to a target nucleic acid having a specific base sequence, comprising 1 to 30 PPR motifs represented by formula 1 below, wherein the A6 amino acid of the first PPR motif from the N-terminus (M1) is a hydrophilic amino acid. [ka] (In the formula: Helix A is a 12-amino acid length region capable of forming an α-helix structure, represented by Equation 2. [ka] In formula 2, A1~A 12 Each of these independently represents an amino acid; X is either absent or a portion consisting of 1 to 9 amino acids in length; Helix B is a region consisting of 11-13 amino acids that can form an α-helix structure; L is the portion represented by formula 3, which is 2 to 7 amino acids long; [ka] In Equation 3, each amino acid is numbered from the C-terminus as "i" (-1), "ii" (-2), and so on. However, L iii ~L vii (It may not exist.) [6] The protein according to 5, wherein the A9 amino acid of M1 is a hydrophilic amino acid or glycine. [7] The protein according to 5 or 6, wherein the A6 amino acid of M1 is asparagine or aspartic acid. [8] The protein described in any one of items 5-7, wherein the A9 amino acid of M1 is glutamine, glutamic acid, lysine, or glycine. [9] A protein described in any one of items 5 to 8, wherein the A6 amino acid of M1 and the A9 amino acid of M1 are in any of the following combinations. • The combination in which amino acid A6 is asparagine and amino acid A9 is glutamic acid. • The combination in which amino acid A6 is asparagine and amino acid A9 is glutamine. • A combination where amino acid A6 is asparagine and amino acid A9 is lysine. • The combination where amino acid A6 is aspartic acid and amino acid A9 is glycine.

[10] A fusion protein comprising at least one selected from the group consisting of a fluorescent protein, a nuclear localization signal peptide, and a tag protein, and a PPR protein containing the PPR motif described in 1 or 2 as the first PPR motif from the N-terminus, or a protein described in any one of 5 to 9.

[11] A method for modifying a PPR protein that can bind to a target nucleic acid having a specific base sequence, including the PPR motif defined in 3, wherein the A6 amino acid of the first PPR motif from the N-terminus (M1) is made more hydrophilic.

[12] A method for detecting nucleic acids, characterized by using a PPR protein containing the PPR motif described in 1 or 2 as the first PPR motif from the N-terminus, a protein described in any one of items 5 to 9, or a fusion protein described in 10.

[13] A nucleic acid encoding a PPR motif as described in 1 or 2, a PPR protein containing the PPR motif described in 1 or 2 as the first PPR motif from the N-terminus, or any one of the proteins described in 5 to 9.

[14] A vector containing the nucleic acid described in 13.

[15] Cells (excluding human individuals) containing the vector described in 14.

[16] A method for manipulating nucleic acids (excluding implementation in human organisms) using the PPR motif described in 1 or 2, a PPR protein containing the PPR motif described in 1 or 2 as the first PPR motif from the N-terminus, a protein described in any one of items 5 to 9, or a vector described in 14.

[17] A method for producing an organism, including the operation described in 16. [Brief explanation of the drawing]

[0010] [Figure 1] PPR motif design method. A: The 6th and 9th amino acids of the first motif are exposed on the outside. B: For the 6th and 9th amino acids of the first motif that recognize cytosine, the following combinations were selected: leucine and glycine (C6L9G) as a typical combination, and leucine and glutamate (C6L9E), asparagine and glutamine (C6N9Q), asparagine and glutamate (C6N9E), asparagine and lysine (C6N9K), and aspartate and glycine (C6D9G) as variants. [Figure 2] Aggregation and nuclear translocation of each PPR protein. Intracellular expression of PPRs fused with GFP and nuclear localization signal sequences was confirmed using fluorescence microscopy. When fused with EGFP, PPRcag1 (6L9G) and PPRcag2 (6L9E) did not localize to the nucleus but strongly aggregated around it. On the other hand, PPRcag3 (6N9Q), PPRcag4 (6N9E), PPRcag5 (6N9K), and PPRcag6 (6D9G) showed low aggregation but did not localize to the nucleus. When fused with mClover3, PPRcag1 (6L9G) and PPRcag2 (6L9E) localized to the nucleus but aggregated within the nucleus. PPRcag3 (6N9Q), PPRcag4 (6N9E), PPRcag5 (6N9K), and PPRcag6 (6D9G) were localized to the nucleus and did not exhibit aggregation. [Figure 3]Binding experiments between PPR proteins and RNA were conducted. It was found that all PPRs, including those with amino acid mutations at positions 6 and 9, specifically bound to the target CAGx6. The binding affinity to the target sequence was similar for PPRcag2 compared to PPRcag1, approximately 80% for PPRcag3, approximately 60% for PPRcag4, approximately 120% for PPRcag5, and approximately 130% for PPRcag6. [Figure 4] The effect of the N-terminal to the first PPR motif on aggregation. Each PPR protein was prepared and purified in an E. coli expression system and separated by gel filtration chromatography. A smaller elution volume indicates a larger molecular size. In V2, elution occurred in fractions of 8 to 10 mL, while in v3.2, a peak was observed in fractions of 12 to 14 mL. This suggests that aggregation may be occurring in v2 due to its larger protein size, and that this aggregation was improved in v3.2. [Modes for carrying out the invention]

[0011] [PPR motif, PPR protein] (definition) In this invention, when referring to a PPR motif, unless otherwise specified, it means a polypeptide composed of 30 to 38 amino acids whose E value obtained at PF01535 in Pfam and PS51375 in Prosite when the amino acid sequence is analyzed using a web-based protein domain search program is less than or equal to a predetermined value (preferably E-03). The positional numbers of the amino acids constituting the PPR motif as defined in this invention are almost synonymous with PF01535, but correspond to the number obtained by subtracting 2 from the amino acid position in PS51375 (e.g., the 1st amino acid in this invention → the 3rd amino acid in PS51375). However, when referring to the "ii" (-2) amino acid, it refers to the second amino acid from the end (C-terminus) of the amino acid constituting the PPR motif, or the -2 amino acid, two positions N-terminus relative to the 1st amino acid of the next PPR motif. If the next PPR motif is not clearly identified, the amino acid two positions prior to the 1st amino acid of the next helix structure is referred to as "ii". For information on Pfam, please refer to http: / / pfam.sanger.ac.uk / , and for information on Prosite, please refer to http: / / www.expasy.org / prosite / .

[0012] The conserved amino acid sequence of the PPR motif has low conservation at the amino acid level, but the two α-helices are well conserved in the secondary structure. A typical PPR motif consists of 35 amino acids, but its length is variable, ranging from 30 to 38 amino acids.

[0013] More specifically, the PPR motif in this invention consists of a polypeptide with a length of 30 to 38 amino acids, represented by Formula 1.

[0014] [ka] During the ceremony: Helix A is a 12-amino acid length region capable of forming an α-helix structure, represented by Equation 2.

[0015] [ka] In Formula 2, A1 to A 12 each independently represents an amino acid; X is a part that is absent or consists of 1 to 9 amino acids in length; Helix B is a part that consists of 11 to 13 amino acids in length and can form an α - helix structure; L is a part represented by Formula 3 and consists of 2 to 7 amino acids in length;

[0016] [Chemical Formula] In Formula 3, each amino acid is numbered from the C - terminal side as "i" (-1), "ii" (-2), etc. However, L iii ~L vii may not exist.

[0017] When referring to a PPR protein in the present invention, unless otherwise specified, it refers to a PPR protein having one or more, preferably two or more of the above - mentioned PPR motifs. When referring to a protein in this specification, unless otherwise specified, it refers to all substances composed of polypeptides (chains in which multiple amino acids are peptide - bonded), including those composed of relatively low - molecular - weight polypeptides. When referring to an amino acid in the present invention, it may usually refer to a normal amino acid molecule, or may refer to an amino acid residue constituting a peptide chain. Which one it refers to is clear to those skilled in the art from the context. ​​​​​​​​​​In the PPR motif, the combination of three amino acids, positions 1, 4, and ii, is important for specific binding to a base, and this combination allows us to determine which base will bind (see Patent Documents 1 and 2 above).

[0021] Specifically, regarding RNA-binding PPR motifs, the relationship between the combination of amino acids 1, 4, and ii and the bases to which they can bind is as follows (see Patent Document 1 above). (3-1) A1, A4, and L ii When the combination of these three amino acids is valine, asparagine, and aspartic acid, the PPR motif has selective RNA base binding ability, strongly binding to U, then to C, and then to A or G. (3-2) A1, A4, and L ii In the case of the three amino acid combinations, valine, threonine, and asparagine, respectively, the PPR motif has selective RNA base binding ability, binding strongly to A, then to G, then to C, but not to U. (3-3) A1, A4, and L ii The combination of these three amino acids, in order, valine, asparagine, and asparagine, respectively, has a selective RNA base-binding ability in which its PPR motif strongly binds to C, then to A or U, but does not bind to G. (3-4) A1, A4, and L ii When the combination of these three amino acids is glutamic acid, glycine, and aspartic acid, the PPR motif has selective RNA base binding ability, strongly binding to G but not to A, U, and C. (3-5) A1, A4, and L ii The combination of these three amino acids, in order, isoleucine, asparagine, and in the case of asparagine, its PPR motif has selective RNA base binding ability, binding strongly to C, then to U, then to A, but not to G. (3-6) A1, A4, and L iiWhen the combination of these three amino acids is valine, threonine, and aspartic acid, the PPR motif has selective RNA base binding ability, strongly binding to G, then to U, but not to A and C. (3-7) A1, A4, and L ii When the combination of these three amino acids is lysine, threonine, and aspartic acid, the PPR motif has selective RNA base binding ability, strongly binding to G, then to A, but not to U and C. (3-8) A1, A4, and L ii When the combination of these three amino acids is phenylalanine, serine, and asparagine, the PPR motif has selective RNA base binding ability, binding strongly to A, then to C, and then to G and U. (3-9) A1, A4, and L ii When the combination of these three amino acids is valine, asparagine, and serine, the PPR motif has selective RNA base binding ability, strongly binding to C, then to U, but not to A and G. (3-10) A1, A4, and L ii When the combination of these three amino acids is phenylalanine, threonine, and asparagine, the PPR motif has selective RNA base binding ability, strongly binding to A but not to G, U, and C. (3-11) A1, A4, and L ii When the combination of these three amino acids is, in order, isoleucine, asparagine, and aspartic acid, its PPR motif exhibits selective RNA base binding ability, strongly binding to U, then to A, but not to G and C. (3-12) A1, A4, and L ii When the combination of these three amino acids is threonine, threonine, and asparagine, the PPR motif has selective RNA base binding ability, strongly binding to A but not to G, U, and C. (3-13) A1, A4, and Lii When the combination of these three amino acids is, in order, isoleucine, methionine, and aspartic acid, its PPR motif exhibits selective RNA base binding ability, strongly binding to U, then to C, but not to A and G. (3-14) A1, A4, and L ii The combination of these three amino acids, in the case of phenylalanine, proline, and aspartic acid, is called PPR, and its motif has selective RNA base binding ability, strongly binding to U, then to C, but not to A and G. (3-15) A1, A4, and L ii When the combination of these three amino acids is tyrosine, proline, and aspartic acid, the PPR motif has selective RNA base binding ability, strongly binding to U but not to A, G, and C. (3-16) A1, A4, and L ii When the combination of these three amino acids is leucine, threonine, and aspartic acid, the PPR motif has selective RNA base binding ability, strongly binding to G but not to A, U, and C.

[0022] Specifically, regarding DNA-binding PPR motifs, the relationship between the combination of amino acids 1, 4, and ii and the bases to which they can bind is as follows (see Patent Document 2 cited above). (2-1) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, glycine, and aspartic acid, the PPR motif selectively binds to G; (2-2) A1, A4, and L ii When the combination of the three amino acids is glutamic acid, glycine, and aspartic acid, in that order, the PPR motif selectively binds to G; (2-3) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, glycine, and asparagine, the PPR motif selectively binds to A; (2-4) A1, A4, and L ii When the combination of the three amino acids is glutamic acid, glycine, and asparagine, in that order, the PPR motif selectively binds to A; (2-5) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, glycine, and serine, the PPR motif selectively binds to A, and then to C; (2-6) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, isoleucine, and any amino acid, the PPR motif selectively binds to T and C; (2-7) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, isoleucine, and asparagine, its PPR motif selectively binds to T, and then to C; (2-8) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, leucine, and any amino acid, the PPR motif selectively binds to T and C; (2-9) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, leucine, and aspartic acid, the PPR motif selectively binds to C; (2-10) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, leucine, and lysine, the PPR motif selectively binds to T; (2-11) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, methionine, and any amino acid, the PPR motif selectively binds to T; (2-12) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, methionine, and aspartic acid, the PPR motif selectively binds to T; (2-13) A1, A4, and L ii When the combination of the three amino acids is, in order, isoleucine, methionine, and aspartic acid, its PPR motif selectively binds to T, and then to C; (2-14) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, asparagine, and any amino acid, the PPR motif selectively binds to C and T; (2-15) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, asparagine, and aspartic acid, the PPR motif selectively binds to T; (2-16) A1, A4, and L ii When the combination of the three amino acids is phenylalanine, asparagine, and aspartic acid, respectively, its PPR motif selectively binds to T; (2-17) A1, A4, and L ii When the combination of the three amino acids is glycine, asparagine, and aspartic acid, in that order, the PPR motif selectively binds to T; (2-18) A1, A4, and L ii When the combination of the three amino acids is, in order, isoleucine, asparagine, and aspartic acid, its PPR motif selectively binds to T; (2-19) A1, A4, and L ii When the combination of the three amino acids is threonine, asparagine, and aspartic acid, respectively, its PPR motif selectively binds to T; (2-20) A1, A4, and L ii When the combination of the three amino acids is valine, asparagine, and aspartic acid, respectively, its PPR motif selectively binds to T, and then to C; (2-21) A1, A4, and L iiWhen the combination of the three amino acids is tyrosine, asparagine, and aspartic acid, respectively, its PPR motif selectively binds to T, and then to C; (2-22) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, asparagine, and asparagine, the PPR motif selectively binds to C; (2-23) A1, A4, and L ii When the combination of the three amino acids is isoleucine, asparagine, and asparagine, the PPR motif selectively binds to C; (2-24) A1, A4, and L ii When the combination of the three amino acids is serine, asparagine, and asparagine, the PPR motif selectively binds to C; (2-25) A1, A4, and L ii When the combination of the three amino acids is valine, asparagine, and asparagine, the PPR motif selectively binds to C; (2-26) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, asparagine, and serine, the PPR motif selectively binds to C; (2-27) A1, A4, and L ii When the combination of the three amino acids is valine, asparagine, and serine, in that order, the PPR motif selectively binds to C; (2-28) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, asparagine, and threonine, the PPR motif selectively binds to C; (2-29) A1, A4, and L ii When the combination of the three amino acids is valine, asparagine, and threonine, in that order, the PPR motif selectively binds to C; (2-30) A1, A4, and L iiWhen the combination of the three amino acids is, in order, any amino acid, asparagine, and tryptophan, its PPR motif selectively binds to C, and then to T; (2-31) A1, A4, and L ii When the combination of these three amino acids is, in order, isoleucine, asparagine, and tryptophan, its PPR motif selectively binds to T, and then to C; (2-32) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, proline, and any amino acid, the PPR motif selectively binds to T; (2-33) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, proline, and aspartic acid, the PPR motif selectively binds to T; (2-34) A1, A4, and L ii When the combination of the three amino acids is phenylalanine, proline, and aspartic acid, in that order, the PPR motif selectively binds to T; (2-35) A1, A4, and L ii When the combination of the three amino acids is tyrosine, proline, and aspartic acid, in that order, the PPR motif selectively binds to T; (2-36) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, serine, and any amino acid, the PPR motif selectively binds to A and G; (2-37) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, serine, and asparagine, the PPR motif selectively binds to A; (2-38) A1, A4, and L ii When the combination of the three amino acids is phenylalanine, serine, and asparagine, in that order, the PPR motif selectively binds to A; (2-39) A1, A4, and L iiWhen the combination of the three amino acids is valine, serine, and asparagine, in that order, the PPR motif selectively binds to A; (2-40) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, threonine, and any amino acid, the PPR motif selectively binds to A and G; (2-41) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, threonine, and aspartic acid, the PPR motif selectively binds to G; (2-42) A1, A4, and L ii When the combination of the three amino acids is valine, threonine, and aspartic acid, in that order, the PPR motif selectively binds to G; (2-43) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, threonine, and asparagine, the PPR motif selectively binds to A; (2-44) A1, A4, and L ii When the combination of the three amino acids is phenylalanine, threonine, and asparagine, in that order, the PPR motif selectively binds to A; (2-45) A1, A4, and L ii When the combination of the three amino acids is, in order, isoleucine, threonine, and asparagine, its PPR motif selectively binds to A; (2-46) A1, A4, and L ii When the combination of the three amino acids is valine, threonine, and asparagine, in that order, the PPR motif selectively binds to A; (2-47) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, valine, and any amino acid, the PPR motif binds to A, C, and T, but not to G. (2-48) A1, A4, and L iiWhen the combination of the three amino acids is, in order, isoleucine, valine, and aspartic acid, its PPR motif selectively binds to C, and then to A; (2-49) A1, A4, and L ii When the combination of the three amino acids is, in order, any amino acid, valine, and glycine, the PPR motif selectively binds to C; (2-50) A1, A4, and L ii A protein having selective DNA base-binding ability, determined based on the fact that when the combination of the three amino acids is, in order, any amino acid, valine, and threonine, its PPR motif selectively binds to T.

[0023] (A particularly favorable combination of three amino acids) In RNA-binding PPR motifs, there are representative combinations of the first, fourth, and second amino acids that can recognize and specifically bind to each base. Specifically, the combination that recognizes adenine is valine at the first position, threonine at the fourth position, and asparagine at the second position; the combination that recognizes cytosine is valine at the first position, asparagine at the fourth position, and serine at the second position; the combination that recognizes guanine is valine at the first position, threonine at the fourth position, and aspartic acid at the second position; and the combination that recognizes uracil is valine at the first position, asparagine at the fourth position, and aspartic acid at the second position (see Non-Patent Documents 1-5 above). In one preferred embodiment of the present invention, these combinations are used.

[0024] (Improvement of cohesiveness) The inventors have found, from amino acid information of existing naturally occurring PPR motifs, that the amino acid at the 6th position of the PPR motif is very often hydrophobic (especially leucine), and the amino acid at the 9th position is a non-hydrophilic amino acid (especially glycine). Based on the structures of PPR proteins for which crystal structures have already been obtained (Non-patent Literature 6: Coquille et al., 2014 Nat. Commun.; PDB ID: 4PJQ, 4WN4, 4WSL, 4PJR; Non-patent Literature 7: Shen et al., 2015 Nat. Commun., PDB ID: 5I9D, 5I9F, 5I9G, 5I9H), the inventors hypothesized that the 6th and 9th positions of the first motif (N-terminal side) are exposed to the outside, and that these exposed hydrophobic amino acids cause aggregation (Figure 1A). On the other hand, for the second and subsequent motifs, the 6th and 9th amino acids are embedded within the protein and form a hydrophobic core. Therefore, it was considered that placing hydrophilic residues at the 6th and 9th positions of all motifs could potentially disrupt the protein structure. Thus, in the first motif only, the 6th, and preferably the 6th and 9th amino acids, were made hydrophilic amino acids (asparagine, aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, threonine) to reduce the aggregation of PPR.

[0025] Specifically, do the following: In a protein capable of binding to a target nucleic acid having a specific base sequence, at the first PPR motif from the N-terminus (M1): (1) The A6 amino acid is a hydrophilic amino acid, preferably asparagine or aspartic acid. (2) Furthermore, the A9 amino acid is a hydrophilic amino acid or glycine, preferably glutamine, glutamic acid, lysine, or glycine. (3) Alternatively, the A6 amino acid and A9 amino acid shall be in one of the following combinations: • The combination in which amino acid A6 is asparagine and amino acid A9 is glutamic acid. • The combination in which amino acid A6 is asparagine and amino acid A9 is glutamine. • A combination where amino acid A6 is asparagine and amino acid A9 is lysine. • A combination where amino acid A6 is aspartic acid and amino acid A9 is glycine.

[0026] (New PPR motif) The present invention provides a novel PPR motif with improved aggregation properties, as discovered above, and a novel PPR protein containing the same.

[0027] The novel PPR motifs provided by the present invention are as follows: (C-1) Sequence ID: A PPR motif consisting of one of sequences 4-7; (C-2) Sequence ID: A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 are substituted, deleted, or added in any of the sequences of 4 to 7, and which is cytosine-binding; (C-3) Sequence ID: A PPR motif having at least 80% sequence identity with any one of sequences 4-7, wherein the amino acids at positions 1, 4, 6, and 34 are identical, and which is cytosine-binding; (A-1) A PPR motif consisting of sequence number 8, in which the amino acid at position 6 is substituted with asparagine or aspartic acid; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 in the sequence (A-2)(A-1) are substituted, deleted, or added, and which is adenine-binding; A PPR motif having at least 80% sequence identity with (A-3)(A-1), except that the amino acids at positions 1, 4, 6, and 34 are identical, and which is adenine-binding; (G-1) A PPR motif consisting of sequence number 9, in which the amino acid at position 6 is substituted with asparagine or aspartic acid; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 in the (G-2)(G-1) sequence are substituted, deleted, or added, and which is guanine-binding; A PPR motif having at least 80% sequence identity with (G-3)(G-1), except that the amino acids at positions 1, 4, 6, and 34 are identical, and which is guanine-binding; (U-1) A PPR motif consisting of sequence number 10, in which the amino acid at position 6 is substituted with asparagine or aspartic acid; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 in the (U-2)(U-1) sequence are substituted, deleted, or added, and which is uracil-binding; A PPR motif having at least 80% sequence identity with (U-3)(U-1), except that the amino acids at positions 1, 4, 6, and 34 are identical, and which is uracil-binding.

[0028] Of these PPR motifs, the following are particularly preferred: (C-1) Sequence ID: A PPR motif consisting of one of sequences 4-7; (C-2) Sequence ID: A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 are substituted, deleted, or added in any of the sequences of 4 to 7, and which is cytosine-binding; (C-3) Sequence ID: A PPR motif having at least 80% sequence identity with any one of sequences 4-7, wherein the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is cytosine-binding; (A-1) A PPR motif obtained by substituting the amino acids at positions 6 and 9 in the sequence of sequence number 8 in such a way that one of the combinations defined below is satisfied; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 in the (A-2)(A-1) sequence are substituted, deleted, or added, and which is adenine-binding; A PPR motif having at least 80% sequence identity with (A-3)(A-1), except that the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is adenine-binding; (G-1) A PPR motif consisting of a sequence in which the amino acids at positions 6 and 9 in sequence number 9 are substituted to satisfy one of the combinations defined below; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 in the (G-2)(G-1) sequence are substituted, deleted, or added, and which is guanine-binding; A PPR motif having at least 80% sequence identity with (G-3)(G-1), except that the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is guanine-binding; (U-1) A PPR motif consisting of a sequence in which the amino acids at positions 6 and 9 in sequence number 10 are substituted to satisfy one of the combinations defined below; A PPR motif consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 in the (U-2)(U-1) sequence are substituted, deleted, or added, and which is uracil-binding; A PPR motif having at least 80% sequence identity with (U-3)(U-1), except that the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is uracil-binding. • A combination in which the amino acid at position 6 is asparagine and the amino acid at position 9 is glutamic acid. • A combination where the amino acid at position 6 is asparagine and the amino acid at position 9 is glutamine. • A combination in which the amino acid at position 6 is asparagine and the amino acid at position 9 is lysine. • A combination where the amino acid at position 6 is aspartic acid, and the amino acid at position 9 is glycine.

[0029] The specific sequences for sequence numbers 4-10 are shown in Figure 1 and the sequence listing.

[0030] Among these PPR motifs, the following are even more preferable: (C-4) Sequence number: A PPR motif consisting of a sequence of 4; (A-4) Sequence ID: PPR motif consisting of sequence 58; (G-4) Sequence ID: PPR motif consisting of sequence 59; (U-4) Sequence number: A PPR motif consisting of sequence 60.

[0031] The sequences for sequence numbers 58-60 are shown below and in the sequence listing. Sequence ID: 58 array VTYTTNIDQLCKAGKVDEALELFKEMRSKGVKPNV Sequence ID: 59 array VTYTTNIDQLCKAGKVDEALELFDEMKERGIKPDV Sequence ID: 60 array VTYNTNIDQLCKAGRLDEAEELLEEMEEKGIKPDV

[0032] (PPR protein with improved aggregation properties) The present invention also provides PPR proteins with improved aggregation properties, as found above.

[0033] In one preferred embodiment, the A9 amino acid of M1 is a non-hydrophobic amino acid or glycine, regardless of the other amino acids in M1 or the amino acid sequence of any motif other than M1. The non-hydrophobic amino acid is a hydrophilic amino acid, or cysteine ​​or histidine; preferably a hydrophilic amino acid, namely arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, or threonine; more preferably glutamine, glutamic acid, or lysine.

[0034] In one preferred embodiment, the A9 amino acid of M1 is glutamine, glutamic acid, lysine, or glycine, regardless of the other amino acids of M1 or the amino acid sequence of any motif other than M1.

[0035] In one preferred embodiment, the A6 amino acid of M1 is a nonhydrophobic amino acid, regardless of the other amino acids in M1 or the amino acid sequence of any motif other than M1. The nonhydrophobic amino acid is, for example, a hydrophilic amino acid, or cysteine ​​or histidine; preferably a hydrophilic amino acid, namely arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, or threonine; more preferably asparagine or aspartic acid.

[0036] In one particularly preferred embodiment, the A6 and A9 amino acids of M1 are, in any case, the other amino acids of M1, and in any case, the amino acid sequence of motifs other than M1, one of the following combinations: • The combination in which amino acid A6 is asparagine and amino acid A9 is glutamic acid. • The combination in which amino acid A6 is asparagine and amino acid A9 is glutamine. • A combination where amino acid A6 is asparagine and amino acid A9 is lysine. • A combination where amino acid A6 is aspartic acid and amino acid A9 is glycine.

[0037] In one preferred embodiment of the RNA-binding protein, the A6 and A9 amino acids of M1 satisfy the above conditions, and at least one, preferably half or more, and more preferably all of the PPR motifs contained therein satisfy one of the following: • If the binding base is cytosine, then A1 is valine, A4 is asparagine, and A ii That is serine. • If the binding base is adenine, then A1 is valine, A4 is threonine, and A ii That is asparagine. • If the binding base is guanine, then A1 is valine, A4 is threonine, and A ii That is aspartic acid. • If the base to be bound is uracil or thymine, then A1 is valine, A4 is asparagine, and A ii That is aspartic acid.

[0038] In one preferred embodiment of the RNA-binding protein, M1 is the novel PPR motif described above.

[0039] In one particularly preferred embodiment, M1 is a PPR motif consisting of one of the following polypeptides: • If the binding base is cytosine, the polypeptide consists of one of the sequences from SEQ ID NOs: 4-7. • If the binding base is adenine, the polypeptide obtained by substituting the amino acids at positions 6 and 9 in the sequence of SEQ ID NO:8 to satisfy one of the combinations defined in the following paragraph. • If the binding base is guanine, the polypeptide obtained by substituting the amino acids at positions 6 and 9 in the sequence of SEQ ID NO:9 to satisfy one of the combinations defined in the following paragraph. • If the binding base is uracil, the polypeptide obtained by substituting the amino acids at positions 6 and 9 in the sequence of SEQ ID NO:10 to satisfy one of the combinations defined in the following paragraph. At least one of the PPR motifs other than M1 is a PPR motif consisting of one of the following polypeptides: • If the binding base is cytosine, the polypeptide consists of the sequence SEQ ID NO:2. • If the binding base is adenine, the polypeptide consists of the sequence SEQ ID NO:8. • If the binding base is guanine, the polypeptide consists of the sequence SEQ ID NO:9. • If the binding base is uracil, the polypeptide consists of the sequence SEQ ID NO:10.

[0040] The combination referred to in the above paragraph is one of the following: • The combination in which amino acid A6 is asparagine and amino acid A9 is glutamic acid. • The combination in which amino acid A6 is asparagine and amino acid A9 is glutamine. • A combination where amino acid A6 is asparagine and amino acid A9 is lysine. • A combination where amino acid A6 is aspartic acid and amino acid A9 is glycine.

[0041] In one particularly preferred embodiment, M1 is a PPR motif consisting of one of the following polypeptides: • If the binding base is cytosine, the polypeptide consists of the sequence SEQ ID NOs:4. • If the binding base is adenine, the polypeptide consists of the sequence SEQ ID NO: 58. • If the binding base is guanine, the polypeptide consists of the sequence SEQ ID NO: 59. • If the binding base is uracil, the polypeptide consists of the sequence SEQ ID NO:60. At least one of the PPR motifs other than M1 is a PPR motif consisting of one of the following polypeptides: • If the binding base is cytosine, the polypeptide consists of the sequence SEQ ID NO:2. • If the binding base is adenine, the polypeptide consists of a sequence in which the amino acid at position 15 of the sequence with SEQ ID NO:8 has been substituted with lysine. • If the binding base is guanine, the polypeptide consists of the sequence SEQ ID NO:9. • If the binding base is uracil, the polypeptide consists of the sequence SEQ ID NO:10.

[0042] (Utilization of a high-performance PPR motif skeleton) In one preferred embodiment of the present invention, in the PPR motifs for cytosine, adenine, guanine, and uracil (or thymine), amino acids other than those at positions 1, 4, 6, 9, and ii can be specified as particular amino acids. Specifically, PPR motif sequences from Arabidopsis thaliana in which the amino acid combinations at positions 1, 4, and ii are VTN for adenine-recognizing PPR motifs, VSN for cytosine-recognizing PPR motifs, VTD for guanine-recognizing PPR motifs, and VND for uracil-recognizing PPR motifs are collected, and the types and number of amino acids appearing at each position are compiled, the performance of the PPR motif can be enhanced by selecting amino acids that appear frequently at each position.

[0043] From the perspective of considering amino acids other than the 1st, 4th, 6th, 9th, and iith positions as frequently occurring amino acids as described above, the amino acid sequence of the PPR motif shown below can be used as a reference to obtain RNA-binding PPR proteins. The PPR motif corresponding to cytosine is a PPR motif consisting of one of the sequence IDs: 4-7; The PPR motif corresponding to adenine is a PPR motif consisting of one of the sequences with SEQ ID NO: 8; The PPR motif corresponding to guanine is a PPR motif consisting of one of the sequences with SEQ ID NO:9; The PPR motif corresponding to uracil is a PPR motif consisting of one of the sequences with SEQ ID NO:10.

[0044] (Explanation of terms, etc.) In this invention, when "identity" is used with respect to a base sequence (sometimes called a nucleotide sequence) or amino acid sequence, unless otherwise specified, it refers to the percentage of matching bases or amino acids shared between two sequences when the two sequences are aligned in the most optimal manner. That is, identity can be calculated as (number of matching positions / total number of positions) × 100, and can be calculated using commercially available algorithms. Such algorithms are incorporated into the NBLAST and XBLAST programs described in Altschul et al., J.Mol.Biol. 215(1990) 403-410. More specifically, the search and analysis of identity between base sequences or amino acid sequences can be performed using algorithms or programs well known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX, ClustalW). When using a program, the parameters can be appropriately set by those skilled in the art, or the default parameters of each program may be used. The specific methods of these analysis methods are also well known to those skilled in the art.

[0045] In this specification, when referring to a base sequence or amino acid sequence that is identical (or highly identical), unless otherwise specified, it means that there is at least 70%, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97.5% or more, and even more preferably 99% or more identical.

[0046] Furthermore, in the present invention, when referring to a PPR motif or protein as a "substituted, deleted, or added sequence," the number of amino acids substituted, deleted, or added is not particularly limited for any motif or protein, as long as the motif or protein consisting of that amino acid sequence has the desired function, unless otherwise specified. However, it is usually around 1 to 9 or 1 to 4 amino acids, or even more substitutions are possible if the substitutions are with amino acids of similar properties. Means for preparing polynucleotides or proteins relating to such amino acid sequences are well known to those skilled in the art.

[0047] Similar amino acids refer to amino acids with similar physical properties such as hydroxyl, charge, pKa, and solubility. Examples include the following: Hydrophobic amino acids: alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, tyrosine Nonhydrophobic amino acids; arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, cysteine, histidine; Hydrophilic amino acids; arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine; Acidic amino acids: aspartic acid, glutamic acid; Basic amino acids: lysine, arginine, histidine; Neutral amino acids: alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine; Sulfur-containing amino acids: methionine, cysteine; Aromatic ring amino acids: tyrosine, tryptophan, phenylalanine.

[0048] With regard to genes, nucleic acids, polynucleotides, proteins, and motifs, "creation" can be rephrased as "production" or "manufacturing." Furthermore, when creating genes, etc., by combining parts, the term "construction" is sometimes used, but "construction" can also be rephrased as "production" or "manufacturing."

[0049] The PPR motif of the present invention, proteins containing the same, or nucleic acids encoding them can be produced by those skilled in the art using the prior art and the examples described herein.

[0050] [Characteristics and uses of PPR proteins] (Improvement of PPR protein aggregation) PPR proteins produced using the novel PPR motif of the present invention exhibit reduced intracellular aggregation. Those skilled in the art can evaluate the aggregation of PPR proteins by expressing them intracellularly and confirming the presence or absence of aggregation. Confirmation is even easier if the PPR protein is fused with a fluorescent protein before expression. Our studies have shown that appropriate modification of the amino acids in the first motif of the PPR protein improves the intracellular aggregation of the PPR protein and enhances its ability to transfer to the nucleus.

[0051] (binding force) PPR proteins produced using the novel PPR motif of the present invention not only exhibit reduced intracellular aggregation but may also possess RNA binding performance equivalent to or higher than that of PPR proteins produced using existing PPR motifs for the same target RNA. Equivalent means 55% or more, preferably around 75%.

[0052] The binding affinity to target sequences can be evaluated using methods such as EMSA (Electrophoretic Mobility Shift Assay) or Biacore. EMSA utilizes the property that the mobility of nucleic acid molecules changes when a sample bound to a protein and nucleic acid is subjected to electrophoresis compared to a sample without binding. Intermolecular interaction analysis instruments, such as Biacore, allow for reaction kinetic analysis, enabling detailed protein-nucleic acid binding analysis.

[0053] The binding affinity to a target sequence can also be analyzed by providing a solution containing a candidate protein to an immobilized target nucleic acid and detecting or quantifying the protein bound to the target nucleic acid. This method is an application of ELISA (Enzyme-Linked Immuno Sorbent Assay) and is sometimes called RPB-ELISA (RNA-protein binding ELISA). Specifically, the step of providing a solution containing a candidate protein to an immobilized target nucleic acid can be carried out by flowing a solution containing the target binding protein over the target nucleic acid molecule immobilized on a plate. Various existing immobilization methods can be used to immobilize the target nucleic acid molecule; for example, this can be achieved by providing a nucleic acid probe containing a biotin-modified target nucleic acid molecule to a streptavidin-coated well plate. Detailed experimental conditions can be found in the experimental methods described in detail in the Examples section of this invention. In RPB-ELISA, the binding affinity between the target PPR protein and its target RNA can be determined by subtracting the background signal (the luminescence signal value when the target PPR protein is added without the target RNA) from the luminescence amount of a sample to which the target PPR protein and its target RNA have been added.

[0054] [Utilization of PPR protein] (complex, fusion protein) The PPR motifs or PPR proteins provided by the present invention can be formed into complexes by linking functional regions. Furthermore, proteinaceous functional regions can be linked to form fusion proteins. A functional region refers to a portion that has a specific biological function within a living organism or cell, such as enzymatic function, catalytic function, inhibitory function, or enhancement function, or a portion that functions as a label. Such regions include, for example, proteins, peptides, nucleic acids, physiologically active substances, and drugs. In the following description of the present invention, fusion proteins may be used as an example in relation to complexes; however, those skilled in the art will be able to understand the case of complexes other than fusion proteins in accordance with this description.

[0055] In one preferred embodiment, the functional domain is a ribonuclease (RNase). Examples of RNases include RNase A (e.g., bovine pancreatic ribonuclease A: PDB 2AAS) and RNase H.

[0056] In one preferred embodiment, the functional domain is a fluorescent protein. Examples of fluorescent proteins include mCherry, EGFP, GFP, Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, CFP, TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, HyPer, TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana, KusabiraOrange, mOrange, TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, TurboFP602, mRFP1, JRed, KillerRed, HcRed, KeimaRed, mRasberry, mPlum, PS-CFP, Dendra2, Kaede, EosFP, and KikumeGR. From the viewpoint of improved aggregation and / or efficient translocation to the nucleus as a fusion protein, a preferred example is mClover3.

[0057] In one preferred embodiment, the functional domain is a functional domain that enhances protein expression from target mRNA when the target is mRNA (WO2017 / 209122). Examples of functional domains that enhance protein expression from mRNA may be, for example, all or part of a functional domain of a protein known to directly or indirectly promote mRNA translation. More specifically, it may be a domain that induces ribosomes to mRNA, a domain related to mRNA translation initiation or promotion, a domain related to mRNA transport out of the nucleus, a domain related to binding to the endoplasmic reticulum membrane, a domain containing an endoplasmic reticulum retention signal (ER retention signal) sequence, or a domain containing an endoplasmic reticulum signal sequence. Even more specifically, the above-mentioned domain that induces ribosomes to mRNA may be a domain containing all or part of a polypeptide selected from the group consisting of DENR (Density-regulated protein), MCT-1 (Malignant T-cell amplified sequence 1), TPT1 (Translationally-controlled tumor protein), and Lerepo4 (Zinc finger CCCH-domain). Furthermore, the domains related to translation initiation or promotion of mRNA may be domains containing all or a functional part of a polypeptide selected from the group consisting of eIF4E and eIF4G. Furthermore, the domains related to the transport of mRNA out of the nucleus may be domains containing all or a functional part of SLBP (Stem-loop binding protein). Furthermore, the domains related to binding to the endoplasmic reticulum membrane may be domains containing all or a functional part of a polypeptide selected from the group consisting of SEC61B, TRAP-alpha (Translocon associated protein alpha), SR-alpha, Dia1 (Cytochrome b5 reductase 3), and p180.Furthermore, the above-mentioned endoplasmic reticulum retention signal (ER) sequence may be a signal sequence containing the KDEL (SEQ ID NO: 55) or KEEL (SEQ ID NO: 56) sequence. Also, the ER signal sequence may be a signal sequence containing MGWSCIILFLVATATGAHS (SEQ ID NO: 57).

[0058] In the present invention, the functional region may be fused to the N-terminal side of the PPR protein, to the C-terminal side, or to both the N-terminal and C-terminal sides. Furthermore, the complex or fusion protein may contain multiple functional regions (for example, 2 to 5). Moreover, in the complex or fusion protein of the present invention, the functional region and the PPR protein may be indirectly fused via a linker or the like.

[0059] (Nucleic acids, vectors, and cells encoding PPR proteins, etc.) The present invention also provides nucleic acids encoding the PPR motif, PPR protein, or fusion protein described above, as well as vectors containing nucleic acids (e.g., vectors for amplification, expression vectors). Vectors for amplification can be used with E. coli or yeast as hosts. In this specification, an expression vector means a vector containing, for example, DNA having a promoter sequence, DNA encoding a desired protein, and DNA having a terminator sequence from upstream, but the sequences do not necessarily have to be in this order as long as the desired function is performed. In the present invention, various vectors that are commonly used by those skilled in the art can be rearranged and used.

[0060] The PPR protein or fusion protein of the present invention can function in eukaryotic cells (e.g., animals, plants, microorganisms (yeast, etc.), protists). In particular, the fusion protein of the present invention can function in animal cells (in vitro or in vivo). Examples of animal cells into which the PPR protein or fusion protein of the present invention, or vectors expressing it, can be introduced include cells derived from humans, monkeys, pigs, cattle, horses, dogs, cats, mice, and rats. Examples of cultured cells into which the PPR protein or fusion protein of the present invention, or vectors expressing it, can be introduced include, but are not limited to, Chinese hamster ovary (CHO) cells, COS-1 cells, COS-7 cells, VERO (ATCC CCL-81) cells, BHK cells, canine kidney-derived MDCK cells, hamster AV-12-664 cells, HeLa cells, WI38 cells, 293 cells, 293T cells, and PER.C6 cells.

[0061] (Application) The PPR protein or fusion protein of the present invention may be capable of delivering and activating a functional region in a nucleic acid sequence-specific manner within a living organism or cell. A complex to which a labeled portion such as GFP is linked can be used to visualize a desired RNA in vivo.

[0062] Furthermore, the PPR proteins or fusion proteins of the present invention can be modified or disrupted in a nucleic acid sequence-specific manner within cells or in vivo, and may also be able to confer new functions. In particular, RNA-binding PPR proteins are involved in all RNA processing steps found in organelles, including cleavage, RNA editing, translation, splicing, and RNA stabilization. Therefore, the methods for modifying PPR proteins provided by the present invention, and the PPR motifs and PPR proteins provided by the present invention, are expected to have the following uses in various fields.

[0063] (1) Medical • To produce PPR proteins that recognize and bind to specific RNAs associated with specific diseases. Furthermore, to analyze the target sequence and associated proteins of these specific RNAs. The results of these analyses can be used to search for compounds for the treatment of diseases.

[0064] For example, in animals, abnormalities in a PPR protein identified as LRPPRC are known to cause Leigh syndrome French Canadian (LSFC; subacute necrotizing encephalomyelopathy). The present invention may contribute to the treatment (prevention, therapy, and suppression of progression) of LSFC. Many existing PPR proteins function to designate editing sites for RNA manipulation (transformation of genetic information on RNA; often C→U). This type of PPR protein has an additional motif at the C-terminus that is suggested to interact with RNA editing enzymes. It is expected that PPR proteins with such a structure can be used to introduce nucleotide polymorphisms or to treat diseases or conditions caused by nucleotide polymorphisms.

[0065] • Create cells with controlled RNA suppression and expression. Such cells include stem cells (e.g., iPS cells) with monitored differentiated and undifferentiated states, model cells for evaluating cosmetics, and cells in which the expression of functional RNA can be switched ON / OFF for the purpose of elucidating drug discovery mechanisms and conducting pharmacological tests.

[0066] • To create PPR proteins that specifically bind to specific RNAs associated with specific diseases. By introducing such PPR proteins into cells using plasmids, viral vectors, mRNA, or purified proteins, the PPR proteins can bind to their target RNAs within the cells, thereby altering (improving) the RNA function that causes the disease. Means of altering this function include, for example, changes in RNA structure due to binding, knockdown through degradation, changes in splicing reactions through splicing, and base substitution.

[0067] (2) Agriculture, forestry and fisheries • To improve yield and quality in agricultural products, forestry products, fishery products, etc. • To breed organisms with improved disease resistance, improved environmental tolerance, or enhanced or new functionalities.

[0068] For example, with regard to first-generation hybrid (F1) crops, it may be possible to artificially create F1 crops by using PPR proteins to stabilize mitochondrial RNA and regulate translation, thereby improving yield and quality. RNA manipulation and genome editing using PPR proteins enable more accurate and rapid improvement of biological varieties and breeding (genetically improving organisms) than conventional technologies. Furthermore, unlike genetic modification, which alters traits with foreign genes, RNA manipulation and genome editing using PPR proteins are technologies that deal with the RNA and genomes that are originally present in plants and animals, making them similar to traditional breeding methods such as mutant selection and backcrossing. Therefore, they can reliably and quickly address global food and environmental problems.

[0069] (3)Chemistry In the production of useful substances using microorganisms, cultured cells, plants, and animals (e.g., insects), protein expression levels are controlled by manipulating DNA and RNA. This improves the productivity of useful substances. Examples of useful substances include proteinaceous substances such as antibodies, vaccines, and enzymes, as well as relatively low-molecular-weight compounds such as pharmaceutical intermediates, fragrances, and dyes.

[0070] • Improve the efficiency of biofuel production by modifying the metabolic pathways of algae and microorganisms. [Examples]

[0071] [Example 1: Intracellular analysis of fluorescent protein-fusion PPR proteins] (Design of the motif) The target sequence was CAGCAGCAGCAGCAGCAG (SEQ ID NO:1), which consists of six repeats of the CAG sequence. The base recognized by the PPR motif is determined by the amino acid sequences of the 1st, 4th, and 2nd positions. For the PPR motif that recognizes cytosine, valine was placed at the 1st position, asparagine at the 4th position, and serine at the 2nd position. For the PPR motif that recognizes adenine, valine was placed at the 1st position, threonine at the 4th position, and asparagine at the 2nd position. For the PPR motif that recognizes guanine, valine was placed at the 1st position, threonine at the 4th position, and aspartic acid at the 2nd position. For the PPR motif that recognizes uracil, valine should be placed at the 1st position, asparagine at the 4th position, and aspartic acid at the 2nd position.

[0072] Furthermore, regarding the 6th and 9th amino acids of the first motif that recognizes cytosine (Mutated motif in Figure 1A), a typical combination is leucine and glycine (C 6L9G, PPRcag 1. As shown above, SEQ ID NO:2), as a variant, leucine and glutamic acid (C 6L9E, PPRcag 2. SEQ ID NO:3), asparagine and glutamine (C 6N9Q, PPRcag 3. SEQ ID NO: 4), asparagine and glutamic acid (C 6N9E, PPRcag 4. SEQ ID NO: 5), asparagine and lysine (C 6N9K, PPRcag 5. SEQ ID NO: 6), aspartic acid and glycine (C 6D9G, PPRcag 6. SEQ ID NO:7) was selected (Figure 1B). These PPR motif sequences were arranged to bind to the CAGCAGCAGCAGCAGCAG sequence (SEQ ID NO:1 above) to construct the PPR gene (SEQ ID NOs:11-16). In order to efficiently and accurately link the 18 DNA molecules encoding each PPR motif, the amino acids other than the 1st, 4th, 6th, 9th, and iith positions in the PPR motifs for cytosine, adenine, and guanine were selected as the highly frequently occurring amino acids as described above (SEQ ID NOs:8-9, see Patent Document 1 above).

[0073] (Plasmid creation) Plasmids containing the PPR gene were constructed using the Golden Gate method. More specifically, ten intermediate vectors, Dest-a, b, c, d, e, f, g, h, i, and j, were prepared to be linked seamlessly in sequence. 20 different motifs were then created by inserting each of the ten vectors into one of the 20 motifs (one-motif and two-motif PPR motifs corresponding to A, C, G, and U, and two PPR motifs recognizing each base combination of AA, AC, AG, AU, CA, CC, CG, CU, GA, GC, GG, GU, UA, UC, UG, and UU), resulting in 200 different components.

[0074] Dest-a is gaagacataaactccgtggtcacATACagagaccaaggtctcaGTGGtcacatacatgtcttc(SEQ ID NO:43), Dest-b is gaagacatATACagagaccaaggtctcaGTGGtgacataatgtcttc(SEQ ID NO:44), Dest-c is gaagacatcATACagagaccaaggtctcaGTGGttacatatgtcttc(SEQ ID NO:45), Dest-d is gaagacatacATACagagaccaaggtctcaGTGGttacaatgtcttc(SEQ ID NO:46), Dest-e is gaagacattacATACagagaccaaggtctcaGTGGtgacatgtcttc(SEQ ID NO:47), Dest-f is gaagacattgacATACagagaccaaggtctcaGTGGttaatgtcttc(SEQ ID NO:48), Dest-g is gaagacatgttacATACagagaccaaggtctcaGTGGtcatgtcttc(SEQ ID NO:49), Dest-h is gaagacatggtcacATACagagaccaaggtctcaGTGGtatgtcttc(SEQ ID NO:50), Dest-i is gaagacattggttacATACagagaccaaggtctcaGTGGatgtcttc(SEQ ID NO:51), Dest-j is gaagacatgtggtgacATACagagaccaaggtctcaGTGGtcttc(SEQ ID NO:52) This was prepared by creating it using gene synthesis technology and cloning it into pUC57-kan.

[0075] Dest-a to Dest-j were selected according to the target nucleotide sequence and cloned into a vector using the Golden Gate reaction. The vector used here was designed so that the amino acid sequence of MGNSV (SEQ ID NO: 53) was added to the N-terminus of an 18-linked PPR sequence and to the C-terminus of ELTYNTLISGLGKAGRARDPPV (SEQ ID NO: 54). The correct size of the cloned gene was confirmed, and the sequence of the cloned gene was confirmed by sequencing.

[0076] (Detection of expression within cells) The expression plasmid pcDNA3.1 in animal cultured cells contains a CMV promoter and an SV40 poly A signal sequence, allowing for the insertion of the desired gene between them. To detect PPR protein expression in cells, a PPR protein fused with a fluorescent protein was expressed, and its aggregation and nuclear translocation were analyzed from the fluorescence image. Protein genes fused in the following order from the N-terminus: EGFP, nuclear localization signal sequence, PPR protein, and FLAG epitope tag, were inserted into pcDNA3.1 (SEQ ID NOs: 17-22). Additionally, protein genes fused in the following order from the N-terminus: mClover3, PPR protein, nuclear localization signal sequence, and FLAG epitope tag, were inserted into pcDNA3.1 (SEQ ID NOs: 23-28). Plasmids without PPR were also prepared as a control (SEQ ID NOs: 35-36).

[0077] HEK293T cells are placed in a 10cm dish containing 9 mL of DMEM and 1 mL of FBS, 1 x 10 6 Cells were seeded per well. After culturing at 37°C in a 5% CO2 environment for 2 days, the cells were harvested. The harvested cells were placed in a PLL-coated 96-well plate, with 4 x 10⁶ cells per well. 4 Cells were seeded at cells / well and cultured for 1 day at 37°C in a 5% CO2 environment. 200 ng plasmid DNA, 0.6 μL Fugene®-HD (Promega, E2311), and 200 μL Opti-MEM were mixed and added to all wells, and cultured for 1 day at 37°C in a 5% CO2 environment. After culture, the medium was removed, washed once with 50 μL PBS, then 1 μL Hoechst (1 mg / mL, Dojin Chemical, 346-07951) and 50 μL PBS were added, and the mixture was incubated at 37°C in a 5% CO2 environment for 10 minutes, followed by a single wash with 50 μL PBS. After washing, 50 μL PBS was added, and GFP fluorescence and Hoechst fluorescence images of each well were acquired using a DMi8 (Leica) fluorescence microscope.

[0078] The results are shown in Figure 2. Intracellular expression of PPR fused with EGFP and a nuclear localization signaling sequence was confirmed, and PPRcag 1 (6L9G) and PPRcag In 2(6L9E), it was observed that the substance did not localize to the nucleus but strongly aggregated around it. On the other hand, PPRcag 3 (6N9Q), PPRcag 4(6N9E), PPRcag 5(6N9K), PPRcag In 6(6D9G), although the aggregation was low, it did not localize to the nucleus. When mClover3 was fused, PPRcag 1 (6L9G) and PPRcag In 2(6L9E), although localized to the nucleus, aggregation within the nucleus was observed. PPRcag 3 (6N9Q), PPRcag 4(6N9E), PPRcag 5(6N9K), PPRcag In mutation 6 (6D9G), nuclear localization was observed, but no aggregation was seen. Therefore, it was found that mutations 6N9E, 6N9Q, 6N9K, and 6D9G are good for improving aggregation, and that using mClover3 is better than using EGFP for efficient nuclear localization.

[0079] [Example 2: RNA binding analysis of CAG-binding PPR protein] PPRcag 1. PPRcag 2. PPRcag 3. PPRcag 4. PPRcag 5. PPRcag Regarding point 6, recombinant proteins were created and binding experiments were conducted to confirm binding with the target RNA.

[0080] Protein genes were designed by fusing luciferase to the N-terminus and a 6x histidine tag sequence to the C-terminus of each PPR protein, and these were cloned into E. coli expression plasmids (SEQ ID NOs: 29-34). An Nluc-Hisx6 protein gene without PPR protein was also created as a control (SEQ ID NO: 37).

[0081] The completed plasmid was used to transform *E. coli* strain Rosetta (DE3). These *E. coli* were cultured in 2 mL of LB medium containing 100 μg / mL ampicillin at 37°C for 12 hours, and then OD (Oral Dissociation) was performed. 600 However, when the concentration reached 0.5 to 0.8, the culture medium was transferred to a 15°C incubator and allowed to stand for 30 minutes. Then, 100 μL (final concentration 0.1 mM IPTG) was added and the culture was incubated at 15°C for 16 hours. The E. coli pellet was collected by centrifugation at 5,000 xg, 4°C, for 10 minutes, and 1.5 mL of lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP-40, 1 mM MgCl2, 2 mg / ml lysozyme, 1 mM PMSF, 2 μl DNase) was added, and the mixture was frozen at -80°C for 20 minutes. Cell freeze-stretching was performed at 25°C for 30 minutes with perfusion. Subsequently, centrifugation was performed at 3700 rpm, 4°C, for 15 minutes, and the supernatant (E. coli lysate) containing soluble PPR protein was collected.

[0082] The binding experiment between PPR protein and RNA was performed using the same method as the binding experiment between PPR protein and biotinylated RNA on a streptavidin plate.

[0083] RNA probes were synthesized by biotin modification at the 5' end of 30-nucleotide RNAs (SEQ ID NOs: 38-42, respectively) containing the target CAGx6 sequence, and non-target CGGx6, CUGx6, CCGx6, and D1b (UGGUGUAUCUUGUCUUUA) sequences (SEQ ID NOs: 42, pins 8-25). (Grainer) 2.5 pmol biotinylated RNA probes were added to streptavidin-coated plates (Cat No. 15502, Thermofisher) and reacted at room temperature for 30 minutes. The probes were then washed with probe washing buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT, 0.1% BSA). For background measurement, wells with lysis buffer added without biotinylated RNA were also prepared (-Probe). Then, blocking buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT, 1% BSA) was added, and the plate surface was blocked at room temperature for 30 minutes. Then, 1.5 x 10 8 100 μL of E. coli lysate containing a luciferase-fused PPR protein with a luminescence value of LU / μL was added, and the binding reaction was carried out at room temperature for 30 minutes. Subsequently, the wells were washed five times with 200 μL of washing buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT). 40 μL of luciferase substrate (Promega, E151A), diluted 2500-fold with the washing buffer, was added to the wells and reacted for 5 minutes. After reaction, the luminescence was measured using a plate reader (PerkinElmer, Cat No. 5103-35).

[0084] The results are shown in Figure 3. It was found that all PPRs specifically bound to the target, CAGx6. The binding affinity to the target sequence was PPRcag PPRcag compared to 1 2 is about the same, PPRcag 3 is about 80%, PPRcag 4 is about 60%, PPRcag 5 is approximately 120%, PPRcag 6 was approximately 130%. From these, PPRcag It was found that, with the exception of 4, there were almost no changes in binding performance due to mutations.

[0085] [Example 3: Control of PPR protein aggregation] PPR proteins using the V2 motif (nucleotide sequence SEQ ID NO:61, amino acid sequence SEQ ID NO:62) and PPR proteins using the v3.2 motif (nucleotide sequence SEQ ID NO:63, amino acid sequence SEQ ID NO:64) were prepared and purified in an E. coli expression system and separated by gel filtration chromatography. The v2 motif refers to a PPR motif having the sequence SEQ ID NO:2, SEQ ID NOs:8-10, while the v3.2 motif refers to a PPR motif having the sequence SEQ ID NO:4, SEQ ID NOs:58-60 if it is the first from the N-terminus, or if it is the other case, the 15th aspartic acid in SEQ ID NO:8 is substituted with lysine for adenine, and for bases other than adenine, it refers to a PPR motif having a sequence selected from SEQ ID NOs:2,9, or10.

[0086] (Protein expression and purification) E. coli Rosetta strain was transformed using a pE-SUMOpro Kan plasmid containing the DNA sequence encoding the target PPR. After culturing at 37°C, the temperature was lowered to 20°C when the OD600 reached 0.6, and IPTG was added to a final concentration of 0.5 mM to express the target PPR protein as a SUMO-fused protein within the E. coli. After culturing overnight, the cells were collected by centrifugation and resuspended in Lysis Buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl). The E. coli were disrupted by sonication, and after centrifugation at 17000 g for 30 min, the supernatant fraction was transferred to a Ni-Agarose column. After washing the column with Lysis Buffer containing 20 mM imidazole, the SUMO-fused target PPR protein was eluted with Lysis Buffer containing 400 mM imidazole. After elution, the SUMO protein was separated from the target PPR protein using Ulp1, and the protein solution was replaced with ion exchange buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl) by dialysis. Subsequently, cation exchange chromatography was performed using an SP column. After column preparation, the protein was eluted by gradually increasing the NaCl concentration from 200 mM to 1 M. The fraction containing the target PPR protein was finally purified by gel filtration chromatography using a Superdex 200 column. The target PPR protein eluted from ion exchange was prepared by preparing a gel filtration column equilibrated with gel filtration buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP)). Finally, the fraction containing the target PPR protein was concentrated, frozen in liquid nitrogen, and stored at -80°C until use in the next analysis.

[0087] (Gel filtration chromatography) Purified recombinant PPR protein was adjusted to a concentration of 1 mg / ml. Gel filtration chromatography was performed using a Superdex 200 increase 10 / 300 GL (GE Healthcare) system. The adjusted protein was supplied to a gel filtration column equilibrated with 25 mM HEPES pH 7.5, 200 mM NaCl, and 0.5 mM tris(2-carboxyethyl) phosphine (TCEP). The properties of the protein were analyzed by measuring the absorbance of the solution eluted from the gel filtration column at 280 nm.

[0088] (result) The results are shown in Figure 4. A smaller elution volume indicates a larger molecular size. In V2, elution occurred in elution fractions of 8 to 10 mL, while in v3.2, a peak was observed in elution fractions of 12 to 14 mL. This suggests that aggregation may have occurred in v2 due to the larger protein size, and that this aggregation was improved in v3.2.

Claims

1. A composition for treating a disease, comprising a vector containing nucleic acids that encode proteins, The aforementioned protein is given by the following formula 1 【Chemistry 1】 (In the formula: Helix A is a 12-amino acid length region capable of forming an α-helix structure, represented by Equation 2. 【Chemistry 2】 In formula 2, A 1 ~A 12 Each of these independently represents an amino acid; X is either absent or a portion consisting of 1 to 9 amino acids in length; Helix B is a region consisting of 11 to 13 amino acids that can form an α-helix structure; L is the portion represented by formula 3, which is 2 to 7 amino acids long; 【Transformation 3】 In Equation 3, each amino acid is numbered from the C-terminus as "i" (-1), "ii" (-2), and so on. However, L iii ~L vii (It may not exist.) A protein that contains 1 to 30 PPR motifs represented by , and is capable of binding to a target nucleic acid having a specific base sequence. The first PPR motif from the N-terminus (M 1 )but: A 6 A polypeptide in which the amino acid is a hydrophilic amino acid; or One polypeptide listed in Group A and Group B below A composition. Group A: (C-1) Sequence ID: A polypeptide consisting of one of the sequences 4 to 7; (C-2) Sequence ID: A polypeptide consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 are substituted, deleted, or added in any one of sequences 4 to 7, and which is cytosine-binding; (C-3) Sequence ID: A polypeptide having at least 70% sequence identity with any one of sequences 4-7, wherein the amino acids at positions 1, 4, 6, and 34 are identical, and which is cytosine-binding; (A-1) A polypeptide consisting of sequence number 8, wherein the amino acid at position 6 is substituted with asparagine or aspartic acid; A polypeptide having a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 in the sequence of (A-2)(A-1) are substituted, deleted, or added, and which is adenine-binding; A polypeptide having at least 70% sequence identity with (A-3)(A-1), wherein the amino acids at positions 1, 4, 6, and 34 are identical, and which is adenine-binding; (G-1) A polypeptide consisting of sequence number 9, wherein the amino acid at position 6 is substituted with asparagine or aspartic acid; A polypeptide having a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 in the (G-2)(G-1) sequence are substituted, deleted, or added, and which is guanine-binding; A polypeptide having at least 70% sequence identity with (G-3)(G-1), wherein the amino acids at positions 1, 4, 6, and 34 are identical, and which is guanine-binding; (U-1) Sequence ID: A polypeptide consisting of 10 sequences, wherein the amino acid at position 6 is substituted with asparagine or aspartic acid; A polypeptide having a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, and 34 in the (U-2)(U-1) sequence are substituted, deleted, or added, and which is uracil-binding; A polypeptide having at least 70% sequence identity with (U-3)(U-1), wherein the amino acids at positions 1, 4, 6, and 34 are identical, and which is uracil-binding. Group B: (C-1) Sequence ID: A polypeptide consisting of one of the sequences 4 to 7; (C-2) Sequence ID: A polypeptide consisting of a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 are substituted, deleted, or added in any one of sequences 4 to 7, and which is cytosine-binding; (C-3) Sequence ID: A polypeptide having at least 70% sequence identity with any one of sequences 4-7, wherein the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is cytosine-binding; (A-1) A polypeptide obtained by substituting the amino acids at positions 6 and 9 in the sequence of sequence number 8 to satisfy one of the combinations defined below; A polypeptide having a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 are substituted, deleted, or added in the sequence of (A-2)(A-1), and which is adenine-binding; A polypeptide having at least 70% sequence identity with (A-3)(A-1), wherein the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is adenine-binding; (G-1) A polypeptide consisting of a sequence in which the amino acids at positions 6 and 9 in the sequence of sequence number 9 are substituted to satisfy one of the combinations defined below; A polypeptide having a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 in the (G-2)(G-1) sequence are substituted, deleted, or added, and which is guanine-binding; A polypeptide having at least 70% sequence identity with (G-3)(G-1), wherein the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is guanine-binding; (U-1) A polypeptide consisting of a sequence in which the amino acids at positions 6 and 9 in sequence number 10 are substituted to satisfy one of the combinations defined below; A polypeptide having a sequence in which 1 to 9 amino acids other than those at positions 1, 4, 6, 9, and 34 in the (U-2)(U-1) sequence are substituted, deleted, or added, and which is uracil-binding; A polypeptide having at least 70% sequence identity with (U-3)(U-1), except that the amino acids at positions 1, 4, 6, 9, and 34 are identical, and which is uracil-binding. - A combination in which the amino acid at position 6 is asparagine and the amino acid at position 9 is glutamic acid. - A combination in which the amino acid at position 6 is asparagine and the amino acid at position 9 is glutamine. - A combination in which the amino acid at position 6 is asparagine and the amino acid at position 9 is lysine. - A combination where the amino acid at position 6 is aspartic acid, and the amino acid at position 9 is glycine.

2. M 1 A 9 The composition according to claim 1, wherein the amino acid is a hydrophilic amino acid or glycine.

3. M 1 wherein A 6 is asparagine or aspartic acid, the composition according to claim 1 or 2.

4. M 1 A 9 The composition according to any one of claims 1 to 3, wherein the amino acid is glutamine, glutamic acid, lysine, or glycine.

5. M 1 A 6 Amino acids, and M 1 A 9 The composition according to any one of claims 1 to 4, wherein the amino acids are any combination of the following: ・A 6 The amino acid is asparagine, and A 9 Combinations where the amino acid is glutamic acid ・A 6 The amino acid is asparagine, and A 9 Combinations where the amino acid is glutamine ・A 6 The amino acid is asparagine, and A 9 Combinations where the amino acid is lysine ・A 6 It is the amino acid aspartic acid, and A 9 Combinations where the amino acid is glycine

6. M 1 However, the composition according to any one of claims 1 to 5 is any one of the following: (C-4) Sequence ID: A polypeptide consisting of sequence 4; (A-4) Sequence ID: A polypeptide consisting of 58 sequences; (G-4) Sequence ID: A polypeptide consisting of 59 sequences; (U-4) Sequence ID: A polypeptide consisting of sequence 60.

7. A composition for manipulating nucleic acids, comprising a vector containing a nucleic acid encoding a protein as defined in any one of claims 1 to 6.

8. The composition according to any one of claims 1 to 7, wherein the nucleic acid encoding the protein is a nucleic acid encoding a fusion protein containing the protein.

9. The composition according to claim 8, wherein the fusion protein containing the protein is a fusion protein formed by linking the protein with a functional region capable of acting on RNA.

10. The composition according to claim 9, wherein the functional region has an enzymatic function, a catalytic function, an inhibitory function, or an enhancing function.

11. The composition according to claim 9, wherein the functional domain is a ribonuclease.

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