Hybrid Reverse Transcriptases for Long mRNA Sequencing

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Solution Overview

Problem

Current reverse transcriptases, particularly of retroviral origin, face limitations such as low processivity, requirement for specific primers, and inability to transcribe through regions with secondary structure, hindering their utility in applications like single cell transcriptomics and long mRNA sequencing.

Innovation Solution

Development of hybrid reverse transcriptases comprising a non-retroviral retrotransposon covalently joined to a nucleic acid binding protein, enabling improved processivity and ability to transcribe through secondary structures by jumping from the 5' end of one template to the 3' end of another, enhancing their binding affinity and stability.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If retroviral-derived reverse transcriptases are used, then RNAse H activity is present to degrade RNA in RNA:DNA duplex, but processivity is low and the enzyme dissociates from RNA template before reaching the 5' end

Engineering Contradiction:

ImproveprocessivityVSAvoidenzyme dissociation distance

Core Design Contradiction:

ReliabilityVSDuration of action of moving object

Solution Approach 1:

The patent combines a retroviral reverse transcriptase with a non-retroviral reverse transcriptase domain to create a hybrid enzyme. This merging of two different reverse transcriptase domains allows the hybrid enzyme to inherit the RNAse H activity of the retroviral component while gaining the high processivity and template binding affinity of the non-retroviral component, thereby resolving the contradiction between maintaining RNA degradation capability and achieving sustained processivity along the RNA template.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The hybrid reverse transcriptase functions as a composite enzymatic system, integrating domains from retroviral and non-retroviral reverse transcriptases. This composite structure allows the enzyme to combine the RNAse H cleavage activity of retroviral enzymes with the high processivity and secondary structure traversal capabilities of non-retroviral enzymes, achieving both reliable RNA degradation and sustained processivity along the template.

Inventive Principle:

Principle #40Composite materials

2Reliability

If retroviral-derived reverse transcriptases are used, then RNAse H activity is present, but the enzyme cannot transcribe through regions of RNA template having secondary structure

Engineering Contradiction:

Improvetranscription capabilityVSAvoidsecondary structure barrier

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent merges a retroviral reverse transcriptase domain with a non-retroviral reverse transcriptase domain to create a hybrid enzyme. The non-retroviral domain contributes the ability to transcribe through secondary structures, while the retroviral domain maintains RNAse H activity. This combination resolves the contradiction by allowing the enzyme to overcome secondary structure barriers while preserving RNA degradation capability.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The hybrid reverse transcriptase acts as a composite enzymatic system that integrates the secondary structure traversal capability of non-retroviral enzymes with the RNAse H activity of retroviral enzymes. This composite nature allows the enzyme to navigate through structured RNA regions that would otherwise block transcription, while maintaining the ability to degrade RNA in the RNA:DNA duplex.

Inventive Principle:

Principle #40Composite materials

3Adaptability or versatility

If retroviral-derived reverse transcriptases are used, then RNAse H activity is present, but the enzyme has limited utility in single cell transcriptomics and long mRNA sequencing

Engineering Contradiction:

Improveapplication utilityVSAvoidtranscription length

Core Design Contradiction:

Adaptability or versatilityVSDuration of action of moving object

Solution Approach 1:

The patent combines retroviral and non-retroviral reverse transcriptase domains to create a hybrid enzyme with extended processivity. This merging allows the enzyme to transcribe long mRNA sequences required for single cell transcriptomics and long mRNA sequencing applications, while retaining the RNAse H activity characteristic of retroviral enzymes, thereby expanding application utility without sacrificing transcription length capability.

Inventive Principle:

Principle #5Merging (Combining)

4Ease of operation

If a primer complementary to the 3' end of template RNA is required, then reverse transcription can initiate, but the enzyme cannot transcribe long sequences of mRNA

Engineering Contradiction:

Improveprimer requirementVSAvoidtranscribed sequence length

Core Design Contradiction:

Ease of operationVSLength of moving object

Solution Approach 1:

The hybrid reverse transcriptase merges the primer binding capability of retroviral enzymes with the extended processivity of non-retroviral enzymes. This allows the enzyme to initiate reverse transcription with standard primers while maintaining the ability to transcribe long mRNA sequences that would otherwise exceed the processivity limit of retroviral enzymes alone, thus resolving the contradiction between ease of initiation and transcription length.

Inventive Principle:

Principle #5Merging (Combining)

Data Source

PatentUS20250101391A1Hybrid reverse transcriptases

Publication Date: 2025.03.27 BIO RAD LABORATORIES INC

AI summary

Hybrid reverse transcriptases are provided that comprise a non-retroviral retrotransposon, or a fragment of the non-retroviral retrotransposon having reverse transcriptase activity, joined to a nucleic acid binding protein. Also provided are methods of using the hybrid reverse transcriptases to prepare a cDNA molecule library.