Mutant Reverse Transcriptase Thermostability Engineering
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Solution Overview
Problem
Conventional reverse transcriptases have low thermostability, leading to inefficient cDNA synthesis at high temperatures due to RNA degradation and mis-priming, which limits their application in biochemical experiments requiring reverse transcription at elevated temperatures.
Innovation Solution
Mutating specific amino acid residues in M-MLV originated reverse transcriptase, such as substituting threonine with leucine at position 306, along with other targeted substitutions, enhances the enzyme's thermostability, allowing stable reverse transcription activity even at high temperatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If reverse transcription is performed at high temperatures to reduce RNA secondary structure formation, then reverse transcription efficiency is improved, but conventional reverse transcriptase loses activity due to low thermostability
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the reverse transcriptase enzyme to alter its thermostability properties. The T306L substitution changes the enzyme's structural parameters, allowing it to maintain catalytic activity at elevated temperatures (up to 70°C) where conventional reverse transcriptases would denature or lose activity, thereby enabling efficient reverse transcription at high temperatures without sacrificing enzyme stability
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions at specific positions (such as T306L and other site-directed mutations) rather than modifying the entire enzyme globally. These localized changes at critical residues improve thermostability while preserving the overall catalytic function and substrate binding capabilities of the reverse transcriptase enzyme
2Stability of the object's composition
If conventional reverse transcriptase is used at high temperatures, then RNA secondary structure formation is reduced, but the enzyme degrades RNA template prematurely through RNase H activity
Solution Approach 1:
The patent applies parameter changes by engineering mutations that specifically modulate the temperature-dependent behavior of RNase H activity. The mutant reverse transcriptase maintains suppressed RNase H activity at high temperatures while preserving DNA polymerase activity, allowing the enzyme to synthesize cDNA efficiently without degrading the RNA template prematurely, thus maintaining both RNA template integrity and cDNA synthesis productivity
3Manufacturing precision
If conventional reverse transcriptase is used, then enzyme simplicity is maintained, but mis-priming errors occur in cDNA synthesis
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations that enhance the enzyme's specificity and priming accuracy. These mutations alter the interaction parameters between the reverse transcriptase and RNA template, reducing mis-priming events and improving cDNA sequence accuracy while maintaining relatively simple enzyme structure through targeted rather than extensive modifications
Data Source
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AI summary
The present invention relates to a reverse transcriptase having improved thermostability, more precisely a mutant reverse transcriptase with improved thermostability by substitution of one or more amino acids selected from the group consisting of the 63rd glutamine (Q63), the 264th lysine (K264), the 295th lysine (K295), the 306th threonine (T306), the 346th glutamic acid (E346), the 408th proline (P408), the 438th histidine (H438), and the 454th asparagin (N454) of the amino acid sequence of M-MLV originated reverse transcriptase represented by SEQ. ID. NO: 1 with other amino acids. The mutant reverse transcriptase of the present invention demonstrates excellent thermostability, compared with the wild type reverse transcriptase. Therefore, it is advantageous to obtain the target cDNA with stable reverse transcription activity even in the presence of RNA that can form the stable secondary structure at a high temperature which is a structural barrier for reverse transcription.