PPR Motif Design for Sequence-Specific RNA Binding
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Solution Overview
Problem
Existing technologies lack the ability to develop protein factors that can specifically bind to RNA sequences due to a lack of understanding of the correlation between amino acid sequences and RNA affinity, limiting the development of RNA-binding proteins.
Innovation Solution
The method involves identifying the specific amino acids in PPR motifs that determine RNA base selectivity, allowing for the design of proteins that can bind to RNA in a sequence-specific manner by manipulating the structure and combination of PPR motifs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PPR proteins are used to bind RNA sequences, then RNA binding capability is achieved, but the correspondence between amino acid sequences and bindable RNA sequences is not well understood
Solution Approach 1:
The invention changes the parameter of amino acid sequence composition in PPR motifs to achieve specific RNA binding. By identifying that combinations of amino acids at positions 1, 4, and 'ii' determine binding specificity to RNA bases (A, U, G, or C), the patent establishes a systematic parameter change approach where specific amino acid substitutions at these positions create predictable binding specificities, thereby resolving the information loss about amino acid-RNA correspondence.
2Reliability
If puf motifs are used for RNA binding, then RNA binding property is achieved, but the number of available motifs is extremely limited
Solution Approach 1:
The invention makes PPR motifs universal by demonstrating that a single PPR motif structure can bind to any of the four RNA bases (A, U, G, C) through appropriate amino acid selection at positions 1, 4, and 'ii'. This multi-functional capability allows one PPR motif design to serve multiple binding specificities, greatly expanding the versatility compared to the limited puf motifs while maintaining reliable RNA binding.
Solution Approach 2:
By systematically varying the amino acid parameters at positions 1, 4, and 'ii' of the PPR motif, the invention creates a combinatorial library of binding specificities. This parameter change strategy generates numerous distinct motif variants that can target different RNA sequences, resolving the limitation of having only a small number of available motifs.
3Adaptability or versatility
If the structure of PPR motifs is manipulated to achieve arbitrary RNA sequence binding, then design flexibility is improved, but the complexity of determining amino acid combinations increases
Solution Approach 1:
The invention segments the PPR motif structure into specific functional positions (1, 4, and 'ii') that independently contribute to RNA base recognition. This segmentation simplifies the design process by allowing researchers to focus on modifying only these critical positions rather than the entire motif structure, thereby reducing the complexity of determining amino acid combinations while maintaining high design flexibility for targeting arbitrary RNA sequences.
Data Source
AI summary
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.