PPR protein causing less aggregation and use of the same
Inventors
Yagi, Yusuke • Imai, Takayoshi • Tamai, Takayuki • Nakamura, Takahiro • Teramoto, Takamasa
Assignees
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Publication Number
US-12559528-B2
Publication Date
2026-02-24
Expiration Date
Abstract
In order to improve aggregation property of a PPR protein, the A6 amino acid of the 1st PPR motif from the N-terminus (M1) is made more hydrophilic. Further, the A9 amino acid of M1 is made to be 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 a PPR motif as M1 motif may have not only improved aggregation property, but also high binding power to a target nucleic acid.
Core Innovation
The invention relates to PPR protein aggregation reduction by providing a PPR motif defined by specific sequences based on SEQ ID NO: 9 or SEQ ID NO: 10, together with substitutions of the amino acids at positions 6 and 9. The permitted amino-acid combinations at position 6 and position 9 include asparagine or aspartic acid at position 6, together with glutamic acid, glutamine, lysine, or glycine at position 9 as defined by the motif combinations.
The first PPR motif from the N-terminus (M1) is modified at A6 and A9 to improve aggregation while maintaining strong nucleic-acid binding. The described variants include substitutions at A6 as asparagine or aspartic acid and at A9 as hydrophilic amino acids or glycine, and these variants are compared with aggregation behavior when fused to EGFP and localized near nuclei.
The problem being solved is PPR protein aggregation, as shown by the comparison of EGFP-fused PPR constructs where the 6L9G and 6L9E variants aggregated strongly near nuclei, while the 6N9Q, 6N9E, 6N9K, and 6D9G variants showed weak or no aggregation. The patent further maintains that nucleic-acid binding specificity to CAGx6 remained specific and largely comparable across the mutants, and aggregation was reduced in the later v3.2 motif compared to v2 as observed by gel filtration chromatography.
Claims Coverage
The partial content includes one independent claim. It covers a class of PPR motifs defined by SEQ ID NO: 9 or SEQ ID NO: 10 with constrained substitutions at positions 6 and 9, and the dependent claims indicate applications directed to aggregation reduction and solid-phase nucleic-acid detection/quantification.
PPR motif with constrained substitutions at positions 6 and 9
A PPR motif is any one of the following PPR motifs: a PPR motif consisting of the sequence of SEQ ID NO: 9 with such substitutions of the amino acids at positions 6 and 9 that any one of the combinations defined below is satisfied; or a PPR motif consisting of the sequence of SEQ ID NO: 10 with such substitutions of the amino acids at positions 6 and 9 that any one of the combinations defined below is satisfied, wherein the conditions for SEQ ID NO: 8-10 are selected from combinations of asparagine at position 6 with glutamic acid, glutamine, or lysine at position 9, and aspartic acid at position 6 with glycine at position 9.
The claim coverage centers on PPR motifs derived from SEQ ID NO: 9 or SEQ ID NO: 10 with substitutions at amino-acid positions 6 and 9 restricted to defined pairs, thereby supporting downstream uses reflected in the dependent claims for aggregation reduction and solid-phase nucleic-acid detection/quantification.
Stated Advantages
Reduced aggregation of PPR protein variants compared with aggregation-prone variants.
Maintained strong nucleic-acid binding, with nucleic-acid binding specificity to CAGx6 remaining specific and largely comparable across mutants.
Documented Applications
Use of the disclosed PPR motif within PPR proteins comprising 1-30 motifs.
Use of the PPR motif in fusion proteins including fluorescent protein, nuclear localization signal peptide, and tag protein.
Methods for nucleic-acid detection and manipulation using PPR-motif proteins, including detection/quantification of protein binding to a solid-phase target nucleic acid.
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