Hybrid Reverse Transcriptase Domain Swapping for Solubility

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Solution Overview

Problem

Current reverse transcriptase enzymes, such as mouse leukemia virus reverse transcriptase (MLVRT) and feline leukemia virus reverse transcriptase (FLVRT), face challenges in terms of solubility and stability, which affect their expression and accuracy in molecular biology applications.

Innovation Solution

The development of hybrid reverse transcriptases by combining specific domains from MLVRT and FLVRT, such as the finger, palm, thumb, connection, and RNase H domains, with a focus on achieving at least 95% identity to specific sequences, and incorporating mutations like L139, D200, N479, D522, F526, H592, L601, E605, and H632 to enhance thermostability, improves solubility and stability.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If current reverse transcriptase enzymes (MLVRT or FLVRT) are used, then the enzyme can perform RNA-to-DNA conversion, but the solubility and stability are poor

Engineering Contradiction:

Improvesolubility and stabilityVSAvoidexpression efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent creates hybrid reverse transcriptase enzymes by combining domains from MLVRT and FLVRT to form composite enzymatic structures. Specifically, the hybrid enzyme comprises the finger and palm domains from MLVRT (amino acids 1-277) linked to the thumb, connection, and RNase H domains from FLVRT (amino acids 278-667), or vice versa. This composite approach allows the hybrid enzyme to inherit favorable solubility and stability properties from one parent enzyme while maintaining catalytic activity from the other, thereby improving overall reliability and expression efficiency.

Inventive Principle:

Principle #40Composite materials

Solution Approach 2:

The patent divides the reverse transcriptase enzyme into distinct functional domains (finger domain, palm domain, thumb domain, connection domain, and RNase H domain) and recombines these segments from different parent enzymes. This segmentation allows for independent optimization of each domain's contribution to solubility, stability, and catalytic activity, enabling the creation of hybrid enzymes with improved overall performance compared to the parent enzymes.

Inventive Principle:

Principle #1Segmentation

2Reliability

If hybrid reverse transcriptases with multiple domain combinations are created, then solubility and stability improve, but the enzyme structure becomes more complex

Engineering Contradiction:

Improvesolubility and stabilityVSAvoidenzyme structure complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent applies local quality by assigning specific domains from MLVRT or FLVRT to specific positions in the hybrid enzyme structure based on their individual functional properties. The finger and palm domains (critical for catalytic activity) may be sourced from one parent enzyme while the thumb and RNase H domains (affecting stability and solubility) are sourced from the other, optimizing each region's contribution to overall enzyme performance without requiring complete redesign of the entire structure.

Inventive Principle:

Principle #3Local quality

3Temperature

If mutations are incorporated to enhance thermostability, then thermal stability improves, but the enzyme sequence diverges from natural sequences

Engineering Contradiction:

ImprovethermostabilityVSAvoidsequence fidelity

Core Design Contradiction:

TemperatureVSManufacturing precision

Solution Approach 1:

The patent applies parameter changes by introducing specific point mutations at defined positions in the hybrid reverse transcriptase sequence to enhance thermostability. These mutations modify amino acid residues at critical positions to strengthen thermal stability while maintaining the overall hybrid domain architecture. The approach balances sequence modification for improved performance with preservation of the fundamental hybrid enzyme structure and function.

Inventive Principle:

Principle #35Parameter changes

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 hybrid reverse transcriptases exhibit improved solubility, stability, and expression levels compared to non-hybrid enzymes, enabling more efficient RNA-to-DNA conversion and thermal stability during molecular biology procedures.

Implementation Method 1

The reverse transcription of RNA thus has many uses... the reverse transcriptase is followed by polymerase chain reaction amplification

Methodology Applied

Scientific EffectReverse transcription: Enzyme

Implementation Method 2

the hybrid reverse transcriptase as described above has at least one mutation corresponding to L139, D200, N479, D522, F526, H592, L601, E605, and H632 in SEQ ID NO:34 that improves thermostability

Methodology Applied

Scientific EffectThermostability enhancement through mutation: Heat Treatment

Data Source

PatentEP3583207B1Novel reverse transcriptases and uses thereof

Publication Date: 2024.10.09 BIO RAD LABORATORIES INC

Figure 1

AI summary

Hybrid reverse transcriptases formed from portions of FLVRT and MLVRT are provided.