mRNA Display Library Crosslinking for High-Throughput Polypeptide Screening
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Solution Overview
Problem
Current methods for screening polypeptides from large libraries for binding affinity to specific targets are not feasible due to the complexity and size of these libraries, as they require individual sequence testing, which is impractical for libraries exceeding 10^6 members.
Innovation Solution
The development of mRNA display libraries, where mRNA molecules are crosslinked to polypeptides through a linking tRNA, allowing for the covalent attachment of polypeptides to mRNA, enabling their display and selection based on target interaction, using modified nucleosides and crosslinkers that react under specific conditions, such as light exposure or chemical addition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If individual sequences from the library are prepared and tested for binding affinity, then binding affinity can be measured, but the screening is not feasible for very large libraries (>10^6 members)
Solution Approach 1:
The patent combines the polypeptide of interest with its encoding mRNA into a single hybrid molecule through covalent linking. This merging allows simultaneous selection of both the polypeptide's binding function and its encoding sequence in a single screening assay, enabling high-throughput screening of large libraries without requiring individual sequence testing
Solution Approach 2:
The patent uses mRNA as an intermediary carrier that links the polypeptide to its encoding information. The mRNA acts as a mediator that allows the polypeptide to be displayed and selected while simultaneously retaining its genetic code, enabling the screening of vast libraries through selection of the mRNA-polypeptide hybrid molecules
2Stability of the object's composition
If mRNA is crosslinked to polypeptides through linking tRNA, then stable mRNA-polypeptide complexes are formed, but the process requires modified nucleosides and specific crosslinking conditions
Solution Approach 1:
The patent incorporates modified nucleosides (such as psoralen-containing nucleosides) into the mRNA sequence during synthesis before the crosslinking step. This preliminary incorporation of reactive groups prepares the mRNA for subsequent crosslinking with the polypeptide, ensuring stable complex formation without requiring complex in-situ modification procedures
Solution Approach 2:
The patent utilizes photochemical parameters (light exposure) to induce crosslinking between the modified nucleosides in mRNA and corresponding amino acids in the polypeptide. By changing the physical parameter of light exposure, the crosslinking reaction is activated, forming stable mRNA-polypeptide complexes under controlled conditions
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables the efficient selection and optimization of polypeptides with desired binding properties by forming stable mRNA-polypeptide complexes, facilitating the screening of vast libraries and in vitro evolution of polypeptides for improved affinities and properties.
Implementation Method 1
the pairing of the anticodon of the linking tRNA and the complementary codon in the mRNA results in crosslinking the tRNA to the mRNA
Implementation Method 2
a chemical reaction can either occur spontaneously or be induced to occur by addition of certain chemicals or, in some cases, by exposure to light
Data Source
AI summary
Provided herein are, inter alia, methods for linking an mRNA molecule to a polypeptide (e.g., a peptide or a protein) by linking the mRNA molecule to a linking amino acid in the polypeptide, or by linking the mRNA molecule to a linking tRNA to which the polypeptide is attached, via reactions not catalyzed by the ribosome, and methods for making polypeptide libraries. Also provided are mRNA-protein complexes and mRNA-tRNA-protein complexes, libraries containing these complexes, and methods of using these complexes.