Thermostable Proofreading Reverse Transcriptase for High-Fidelity RNA Sequencing
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Solution Overview
Problem
Existing reverse transcriptases are error-prone due to the lack of a proofreading domain, and they struggle with efficient reverse transcription at lower temperatures due to RNA secondary structures, limiting their application in molecular biology and diagnostics.
Innovation Solution
Development of thermophilic or hyperthermophilic proofreading reverse transcriptases derived from Archaeal Family-B polymerases, which have been evolved to possess both proofreading activity and the ability to efficiently transcribe RNA templates, including long ones, at high temperatures, using directed evolution techniques.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If mesophilic reverse transcriptases are used, then they can transcribe RNA templates at lower temperatures, but they lack proofreading activity resulting in high error rates
Solution Approach 1:
The patent merges the RNA template binding capability (originally present in reverse transcriptases) with the proofreading domain (originally present in DNA polymerases) to create a chimeric enzyme that possesses both reverse transcription activity and proofreading activity, thereby resolving the contradiction between fidelity and functional simplicity
Solution Approach 2:
The engineered enzyme achieves multi-functionality by combining reverse transcriptase activity with proofreading capability in a single enzyme molecule, allowing it to perform both RNA-to-DNA conversion and error correction functions that were previously separated in different enzymes
2Productivity
If low polymerization temperature is used, then reverse transcriptase can function, but RNA secondary structures form reducing transcription efficiency
Solution Approach 1:
The patent changes the temperature parameter from low (typical for reverse transcriptases) to high (thermophilic range) by engineering the enzyme's thermal stability, thereby enabling reverse transcription to occur at temperatures that prevent RNA secondary structure formation while maintaining enzyme activity
Solution Approach 2:
The engineered enzyme exhibits dynamic adaptability to high temperature conditions, maintaining structural flexibility and catalytic activity at thermophilic temperatures where wild-type reverse transcriptases would denature, thus resolving the temperature-efficiency contradiction
3Adaptability or versatility
If archaeal Family-B polymerases are used, then high thermostability and fidelity are achieved, but they lack activity on RNA templates
Solution Approach 1:
The patent applies inversion by taking a DNA-specific polymerase and modifying it to accept RNA templates, reversing the typical specificity pattern. The engineered enzyme now prefers RNA templates over DNA templates, achieving the opposite of the wild-type archaeal polymerase's DNA specificity while retaining its thermostability and proofreading capabilities
Data Source
AI summary
Embodiments of the disclosure concern methods and compositions related to generation and/or use of proofreading reverse transcriptases, including those that are thermophilic or hyperthermophilic. The disclosure encompasses specific recombinant polymerases and their use. In some embodiments, the polymerases are utilized for RNA sequencing in the absence of generation of a cDNA intermediate.