Engineered Thermophilic Reverse Transcriptase for High-Temperature RNA Processing
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Solution Overview
Problem
Natural reverse transcriptase enzymes are mesophilic, limiting their use to low to moderate temperatures due to RNA secondary structures, and existing engineered enzymes often lose activity at higher temperatures, making them unsuitable for efficient reverse transcription and amplification reactions, especially in small volumes like single cell profiling.
Innovation Solution
Engineered recombinant archeal Family-B polymerases with high thermostability and reverse transcriptase activity, combining the fidelity of DNA polymerases with the ability to efficiently transcribe RNA templates at high temperatures, including the use of a Thermococcus gorgonarius polymerase domain and a nucleic acid binding domain like HU protein for enhanced processivity and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If mesophilic reverse transcriptase enzymes are used, then reverse transcription activity is maintained at moderate temperatures, but the enzymes lose activity at high temperatures and cannot efficiently process RNA templates with stable secondary structures
Solution Approach 1:
The patent applies parameter changes by modifying the thermal stability parameters of the reverse transcriptase enzyme through directed evolution and rational design. Specific amino acid residues were mutated to enhance the enzyme's structural stability at high temperatures while preserving its reverse transcription catalytic activity, enabling operation from 20°C to 80°C
Solution Approach 2:
The patent creates a chimeric enzyme by combining domains from different polymerases - specifically integrating a thermostable DNA polymerase domain (from Thermococcus gorgonarius) with a reverse transcriptase domain. This composite enzyme structure allows the molecule to exhibit both high thermal stability and RNA template binding capability
2Measurement precision
If reaction volume is reduced to nanoliter scale for single cell profiling, then analysis sensitivity is improved, but inhibitory compounds from biological samples become more concentrated and inhibit reverse transcription and amplification reactions
Solution Approach 1:
The patent employs a disposable microfluidic chip system that contains the reaction mixture in a controlled environment. The chip is designed for single-use, preventing contamination and allowing precise control of reagent volumes down to nanoliter scale, thereby maintaining high sensitivity while managing inhibitory compound concentrations through precise dilution control
3Device complexity
If template switching is used to enable one-step RT-PCR, then reaction complexity is reduced, but additional reagents and optimization steps are required
Solution Approach 1:
The patent creates a universal enzyme system where the engineered reverse transcriptase performs multiple functions: it binds to RNA templates, catalyzes reverse transcription to cDNA, and can directly amplify the cDNA in the same reaction tube. This multi-functionality eliminates the need for separate template switching steps and reduces protocol complexity while maintaining ease of use through a single reagent formulation
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
The engineered enzymes maintain high efficiency and accuracy at both moderate and high temperatures, enabling one-step reverse transcription and amplification, reducing the need for template switching and simplifying RT-PCR reactions, while stabilizing nucleic acids against thermal denaturation.
Implementation Method 1
The ability to convert mature mRNA back into cDNA, without the introns present in genomic DNA is critical for obtaining information in a wide variety of biomedical contexts
Implementation Method 2
engineered enzymes that produce cDNA from an RNA template at high temperatures, for example without limitation temperatures >50° C., >55° C., >60° C., >65° C., or higher
Data Source
AI summary
The present disclosure relates generally to engineered nucleic acid processing enzymes and derivatives thereof, compositions and kits comprising the same; and methods of generating and using the same.