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DNA-binding protein using PPR motif and use of said DNA-binding protein

A protein and motif technology, applied in the protein field

Inactive Publication Date: 2019-04-02
FUJIFILM WAKO PURE CHEM CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, in the case of Pumilio protein, which is known as an RNA-binding factor and can encode RNA recognition, there is no report of its binding to DNA (Non-Patent Documents 8 and 9)

Method used

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  • DNA-binding protein using PPR motif and use of said DNA-binding protein
  • DNA-binding protein using PPR motif and use of said DNA-binding protein
  • DNA-binding protein using PPR motif and use of said DNA-binding protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0448] [Example 1: Collection of novel dPPR molecules]

[0449] Known dPPR proteins are only P63, GUN1, pTAC2, GRP23, and DG1 described in the previous patent (the above-mentioned Patent Document 4), and it is difficult to obtain information for the generalization and improvement of artificial nucleic acid binding modules based on PPR technology. Therefore, this time, we decided to screen for PPR proteins with DNA-binding ability to expand dPPR proteins. The genes of the dPPR molecules found by chance so far contain introns, but the rPPR genes hardly contain introns at all. Using this as an indicator, the whole genome sequence of Arabidopsis thaliana, which is a model plant, was analyzed. As a result, there were 42 PPR genes containing two or more introns. In this example, the DNA-binding ability of these 42 potential dPPR molecules was analyzed in an attempt to identify new dPPR molecules.

[0450] (experimental method)

[0451] 1. Construction of dPPR expression vector

...

Embodiment 2

[0474] [Example 2: dPPR motif-specific amino acid sequence analysis]

[0475] Based on the amino acid sequence information of the modules contained in the dPPR protein identified in Example 1, the dPPR motif-specific amino acid sequence was analyzed.

[0476] First, 9 dPPR proteins were selected from the 18 dPPR proteins identified in Example 1 in order to match to some extent the motif numbers of rPPR proteins in the F-test. Specifically, for dPPR proteins, the DNA binding force was compared with OTP80 by T-test, and dPPR proteins were classified into 3 groups of 0.05-0.01, 0.01-0.001, and <0.001 based on the obtained values, and randomly selected from each group 3, so choose 9 kinds. At all positions, compare the occurrence frequency of amino acids in the PPR motifs (recorded in the order of 1, 2, 3...) of the 9 dPPR molecules and the 5 known rPPR molecules in the table below, and try to determine the characteristics of the dPPR protein The location of the active amino aci...

Embodiment 3-1

[0487] [Example 3-1: Establishment of artificial nucleic acid binding module construction method based on dPPR motif-specific amino acid sequence 1]

[0488] In this Example, in order to verify whether the DNA-binding ability of the PPR protein is enhanced by the dPPR-specific amino acid sequence, the DNA-binding ability of the modified rPPR incorporating the dPPR-specific amino acid sequence was investigated. As the basic rPPR, the consensus PPR (cPPR) reported in Non-Patent Document 15 (Coquille et al., 2014, An artificial PPR scaffold for programmable RNA recognition) was used. It should be noted that cPPR is known as an RNA-binding protein (hence it may be expressed as crPPR), but it is not known whether it binds to DNA. The modification of crPPR utilizes Genewiz's gene synthesis. The DNA binding ability of the engineered crPPR was analyzed by the method used in Example 1. It should be noted that the target sequence of crPPR is AAAAAAAA.

[0489] It should be noted that...

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Abstract

The present invention addresses the problem of generalizing and improving a DNA-binding protein in which a PPR is used. Provided is a protein including at least one PPR motif having a structure represented by formula (1), wherein one PPR motif (Mn) included in the protein is a PPR motif having three amino acids of No. 1 A.A., No. 4 A.A., and No. ''ii'' (-2) A.A., in a combination of specific aminoacids corresponding to a target DNA base or a target DNA base sequence, and satisfies at least one selected from the group consisting of the following (a)-(h): (a) No. 7 A.A. in the PPR motif (Mn) isisoleucine (I); (b) No. 9 A.A. in the PPR motif (Mn) is alanine (A); (c) No. 10 A.A. in the PPR motif (Mn) is tyrosine (Y); (d) No. 18 A.A. in the PPR motif (Mn) is lysine (K), arginine (R), or histidine (H); (e) No. 20 A.A. in the PPR motif (Mn) is glutamic acid (E) or aspartic acid (D); (f) No. 29 A.A. in the PPR motif (Mn) is glutamic acid (E) or aspartic acid (D); (g) No. 31 A.A. in the PPR motif (Mn) is isoleucine (I); and (h) No. 32 A.A. in the PPR motif (Mn) is lysine (K), arginine (R), or histidine (H). (helix A)-X-(helix B)-L formula (1).

Description

technical field [0001] The present invention relates to proteins capable of selectively or specifically binding to target DNA bases or DNA sequences. In the present invention, a triangular pentatricopeptide repeat (PPR) motif is utilized. The present invention also relates to a method for improving binding to a target DNA base or DNA sequence in a protein utilizing a PPR motif. The present invention can be applied to genome editing, and is useful in the fields of medicine, agriculture, and the like. Background technique [0002] In recent years, techniques for binding to target sequences using nucleic acid-binding protein factors identified by various analyzes have been established and put into use. By utilizing this sequence-specific binding, intracellular localization analysis of the target nucleic acid (DNA or RNA), removal of the target DNA sequence, or expression regulation (activation or deactivation) of protein-coding genes present downstream thereof can be performe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12P21/02C12N15/09
CPCC07K14/00C07K14/415C12N9/22C12N15/8213C12N15/8216C07K2319/80C12Y301/21004C12N15/90C12P21/02C12N15/09G01N33/68G16B99/00
Inventor 山根昌之中村崇裕八木祐介
Owner FUJIFILM WAKO PURE CHEM CORP
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