High temperature resistant reverse transcriptase mutants and use thereof
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Solution Overview
Problem
Murine leukemia virus reverse transcriptase (M-MLV) enzymes are limited by their instability and reduced activity at high temperatures, making them inefficient for reverse transcription of RNA with complex secondary structures.
Innovation Solution
A reverse transcriptase mutant is engineered with specific mutations at amino acid residues 446, 313, 583, 607, and 221 to enhance thermostability and activity, increasing efficiency by more than 10 times at 58°C compared to the wild type.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the reaction temperature is increased to break RNA secondary structure, then reverse transcription efficiency is improved, but enzyme stability and activity decrease
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence parameters of the M-MLV enzyme through site-directed mutagenesis. Specific residues (e.g., position 168, 183, 188, 218, 223, 446, 583, 607) are mutated to alter the enzyme's thermal properties, enabling it to maintain stability at higher temperatures (50-65°C) while preserving catalytic activity for reverse transcription.
2Reliability
If random mutation is used to improve thermostability, then high activity and thermostability may be screened, but the process becomes complicated and screening difficulty increases
Solution Approach 1:
The patent applies local quality by focusing mutations on specific local regions of the enzyme that are critical for thermal stability. Instead of random genome-wide mutagenesis, site-directed mutagenesis is used to modify specific amino acid positions (e.g., 168, 183, 188, 218, 223, 446, 583, 607) that are predicted to affect thermostability based on structural and sequence analysis, thereby reducing screening complexity while maintaining effectiveness.
Solution Approach 2:
The patent changes the mutation strategy from random to targeted parameter modification. By using bioinformatics predictions and structural analysis to identify specific amino acid positions for mutation, the approach transforms the complex random screening process into a more manageable site-directed mutagenesis workflow, reducing the search space from 10^7 possible mutants to a focused set of targeted variations.
3Productivity
If site-directed mutations to active sites are used to increase enzyme activity, then affinity to substrate is improved, but overall enzyme stability cannot be enhanced
Solution Approach 1:
The patent applies segmentation by separating the optimization goals into distinct functional regions of the enzyme. Active site mutations (e.g., at positions 168, 183, 188) are designed to enhance catalytic activity and substrate affinity, while separate stability-focused mutations (e.g., at positions 218, 223, 446, 583, 607) are introduced to improve thermostability. This segmented approach allows independent optimization of activity and stability without mutual interference.
Data Source
AI summary
The present invention provides a high temperature resistant reverse transcriptase mutant and its use. In particular, the present invention constructs a reverse transcriptase (M-MLV) mutant library, and the mutants with improved thermostability and higher amplification efficiency are finally screened out by stepwise screening. Under high temperature conditions, the reverse transcription efficiency of the reverse transcriptase mutant of the present invention is greatly improved compared to the wild type.