Pumilio Domain Modular Protein Architecture for RNA Binding
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Solution Overview
Problem
Current technologies lack the ability to systematically target and control unmodified RNA sequences in living cells with high specificity and fidelity, limiting the monitoring and manipulation of RNA functions.
Innovation Solution
Development of a modular protein architecture based on the Pumilio domain, comprising four canonical protein modules that can be concatenated to bind arbitrary RNA sequences, allowing for precise targeting and manipulation of RNA molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If exogenous sequences like MS2 or PP7 are inserted into target RNA, then RNA binding protein can deliver reporter or RNA modification enzyme to RNA of interest, but RNA structure and function are modified
Solution Approach 1:
The invention creates a copy of the Pumilio domain binding interface in the engineered protein modules, allowing these modules to recognize and bind to Pumilio binding sites without requiring exogenous sequences. The modular units contain amino acid sequences that replicate the RNA recognition capability of native Pumilio domains, enabling targeted binding to endogenous RNAs through sequence complementarity rather than structural modification
Solution Approach 2:
The invention changes the chemical parameters of the binding interface by using amino acid side chains (arginine, lysine, asparagine, glutamine) to create electrostatic and hydrogen bonding interactions with RNA bases. This allows specific recognition of RNA sequences through programmable amino acid combinations rather than relying on exogenous RNA sequences
2Adaptability or versatility
If modular protein architecture is designed to bind arbitrary RNA sequences, then any RNA sequence can be targeted by varying building blocks, but protein structure complexity increases
Solution Approach 1:
The invention divides the RNA binding protein into discrete modular units, each containing a Pumilio domain-like structure that recognizes a specific RNA base or motif. These segmented modules can be independently designed and then assembled in different combinations to target various RNA sequences, reducing the complexity of designing entirely new proteins for each target
Solution Approach 2:
The invention creates universal binding modules with conserved structural features (beta-strand arrangements, amino acid positioning) that can recognize different RNA sequences. The same basic module design can be applied repeatedly with minor sequence variations to target any RNA, making the system universally applicable rather than requiring unique designs for each target
Data Source
AI summary
A programmable modular protein architecture for RNA binding comprises a set of modules, derived from RNA-binding protein Pumilio, that can be concatenated into chains of varying composition and length. When bound into a chain, each module has a preferred affinity for a specific RNA base. The chains can bind arbitrary RNA sequences with high specificity and fidelity by varying the sequence of modules within the chains. Each module contains at least 6 amino acids, with the amino acids in positions 1 and 5 providing the preferred affinity for the specific base, and the amino acid at position 2 serving as a stacking unit between concatenated modules. The modules may have four canonic forms, each having a preferred affinity for a different base and characterized by the base with which it has affinity, the two amino acids that provide the affinity, and the amino acid that serves as a stacking unit.