METHOD FOR DESIGNING RNA BINDING PROTEIN UTILIZING PPR MOTIF, AND USE THEREOF

摘要

A method for designing a protein capable of binding in an RNA base selective manner or RNA base sequence specific manner is provided. The protein of the present invention is a protein containing one or more of PPR motifs (preferably 2 to 14 PPR motifs) each consisting of a polypeptide of 30- to 38-amino acid length represented by the formula 1 (wherein Helix A is a moiety of 12-amino acid length capable of forming an α-helix structure, and is represented by the formula 2, wherein, in the formula 2, A1 to A12 independently represent an amino acid; X does not exist, or is a moiety of 1- to 9-amino acid length; Helix B is a moiety of 11- to 13-amino acid length capable of forming an α-helix structure; and L is a moiety of 2- to 7-amino acid length represented by the formula 3, wherein, in the formula 3, the amino acids are numbered “i” (−1), “ii” (−2), and so on from the C-terminus side, provided that Liii to Lvii may not exist), and combination of three amino acids A1, A4 and Lii, or combination of two amino acids A4, and Lii is a combination corresponding to a target RNA base or base sequence.

主权项

1. A method for identifying a target base or base sequence for an RNA-binding protein comprising one or more of PPR motifs, the method comprising:
determining presence or absence of a base corresponding to a combination of three amino acids A1, A4 and Lii of the one or more PPR motifs on the basis of any of the propositions (3-1) to (3-16):
(3-1) when the three amino acids A1, A4, and Lii are valine, asparagine, and aspartic acid, respectively, the PPR motif can selectively bind to U (uracil);(3-2) when the three amino acids A1, A4, and Lii are valine, threonine, and asparagine, respectively, the PPR motif can selectively bind to A (adenine);(3-3) when the three amino acids A1, A4, and Lii are valine, asparagine, and asparagine, respectively, the PPR motif can selectively bind to C (cytosine);(3-4) when the three amino acids A1, A4, and Lii are glutamic acid, glycine, and aspartic acid, respectively, the PPR motif can selectively bind to G (guanine);(3-5) when the three amino acids A1, A4, and Lii are isoleucine, asparagine, and asparagine, respectively, the PPR motif can selectively bind to C or U;(3-6) when the three amino acids A1, A4, and Lii are valine, threonine, and aspartic acid, respectively, the PPR motif can selectively bind to G;(3-7) when the three amino acids A1, A4, and Lii are lysine, threonine, and aspartic acid, respectively, the PPR motif can selectively bind to G;(3-8) when the three amino acids A1, A4, and Lii are phenylalanine, serine, and asparagine, respectively, the PPR motif can selectively bind to A;(3-9) when the three amino acids A1, A4, and Lii are valine, asparagine, and serine, respectively, the PPR motif can selectively bind to C;(3-10) when the three amino acids A1, A4, and Lii are phenylalanine, threonine, and asparagine, respectively, the PPR motif can selectively bind to A;(3-11) when the three amino acids A1, A4, and Lii are isoleucine, asparagine, and aspartic acid, respectively, the PPR motif can selectively bind to U or A;(3-12) when the three amino acids A1, A4, and Lii are threonine, threonine, and asparagine, respectively, the PPR motif can selectively bind to A;(3-13) when the three amino acids A1, A4, and Lii are isoleucine, methionine, and aspartic acid, respectively, the PPR motif can selectively bind to U or C;(3-14) when the three amino acids A1, A4, and Lii are phenylalanine, proline, and aspartic acid, respectively, the PPR motif can selectively bind to U;(3-15) when the three amino acids A1, A4, and Lii are tyrosine, proline, and aspartic acid, respectively, the PPR motif can selectively bind to U; and(3-16) when the three amino acids A1, A4, and Lii are leucine, threonine, and aspartic acid, respectively, the PPR motif can selectively bind to Q wherein each PPR motif consists of a polypeptide of 30- to 38-amino acid length represented by the formula 1:
(HelixA)-X-(HelixB)-L  (Formula 1) wherein, in formula 1, Helix A is a moiety of 12-amino acid length capable of forming an α-helix structure, and is represented by the formula 2:
A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12  (Formula 2) wherein, in the formula 2, A1 to A12 independently represent an amino acid; wherein, in the formula 1, X is a moiety of 1- to 9-amino acid length and is optional; wherein, in the formula 1, Helix B is a moiety of 11- to 13-amino acid length capable of forming an α-helix structure; and wherein, in the formula 1, L is a moiety of 2- to 7-amino acid length represented by the formula 3;
Lvii-Lvi-Lv-Liv-Liii-Lii-Li  (Formula 3) wherein, in the formula 3, Li to Lvii independently represent an amino acid, and Liii to Lvii are optional, and wherein a combination of three amino acids A1, A4 and Lii, or a combination of two amino acids A4 and Lii corresponds to a target RNA base or base sequence. 2. A method for identifying a PPR protein that comprises one or more PPR motifs, the method comprising: determining presence or absence of a combination of three amino acids A1, A4 and Lii of the one or more on the basis of any of the propositions (2-1) to (2-12):
(2-1) when A4 and Lii are asparagine and aspartic acid, respectively, the motif can selectively bind to U;(2-2) when A4 and Lii are asparagine and asparagine, respectively, the motif can selectively bind to C;(2-3) when A4 and Lii are threonine and asparagine, respectively, the motif can selectively bind to A;(2-4) when A4 and Lii are threonine and aspartic acid, respectively, the motif can selectively bind to G;(2-5) when A4 and Lii are serine and asparagine, respectively, the motif can selectively bind to A;(2-6) when A4 and Lii are glycine and aspartic acid, respectively, the motif can selectively bind to G;(2-7) when A4 and Lii are asparagine and serine, respectively, the motif can selectively bind to C;(2-8) when A4 and Lii are proline and aspartic acid, respectively, the motif can selectively bind to U;(2-9) when A4 and Lii are glycine and asparagine, respectively, the motif can selectively bind to A;(2-10) when A4 and Lii are methionine and aspartic acid, respectively, the motif can selectively bind to U;(2-11) when A4 and Lii are leucine and aspartic acid, respectively, the motif can selectively bind to C; and(2-12) when A4 and Lii are valine and threonine, respectively, the motif can
selectively bind to U, wherein each PPR motif consists of a polypeptide of 30- to 38-amino acid length represented by the formula 1:
(HelixA)-X-(HelixB)-L  (Formula 1) wherein, in formula 1, Helix A is a moiety of 12-amino acid length capable of forming an α-helix structure, and is represented by the formula 2:
A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12  (Formula 2) wherein, in the formula 2, A1 to A12 independently represent an amino acid; wherein, in the formula 1, X is a moiety of 1- to 9-amino acid length and is optional; wherein, in the formula 1, Helix B is a moiety of 11- to 13-amino acid length capable of forming an α-helix structure; and wherein, in the formula 1, L is a moiety of 2- to 7-amino acid length represented by the formula 3;
Lvii-Lvi-Lv-Liv-Liii-Lii-Li  (Formula 3) wherein, in the formula 3, Li to Lvii independently represent an amino acid, and Liii to Lvii are optional, and wherein a combination of three amino acids A1, A4 and Lii, or a combination of two amino acids A4 and Lii corresponds to a target RNA base or base sequence.