Linker-Bridged Reverse Transcriptase Enzyme for cDNA Synthesis
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Solution Overview
Problem
Current methods for reverse transcription face challenges in efficiently synthesizing cDNA due to the RNase H activity of reverse transcriptase, which degrades mRNA templates, leading to reduced yield and quality of the cDNA product.
Innovation Solution
Development of a linker-bridged gene or domain fusion reverse transcriptase enzyme with modified or deleted RNase H domains, combined with mutated RNase A, to enhance affinity for RNA-primers and RNA-DNA hybrids, thereby reducing template degradation and improving processivity and fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RNase H activity is present in reverse transcriptase, then RNA degradation occurs during reverse transcription, but this leads to reduced cDNA yield and premature termination
Solution Approach 1:
The patent extracts and removes the RNase H domain from the reverse transcriptase enzyme through genetic engineering, creating a mutant RT enzyme that lacks RNase H activity. This is achieved by deleting the RNase H coding sequence from the pol gene, resulting in an enzyme that can synthesize cDNA without simultaneously degrading the RNA template, thereby resolving the contradiction between reliability and quantity
Solution Approach 2:
The patent segments the reverse transcriptase enzyme into separate functional domains: the DNA polymerase domain is retained while the RNase H domain is removed. This segmentation allows the polymerase function to operate independently without the harmful RNase H activity, enabling full-length cDNA synthesis while maintaining enzymatic function
2Productivity
If RNase H degrades mRNA template during first-strand synthesis, then competition occurs between deadnylation and DNA synthesis initiation, but this reduces cDNA product yield
Solution Approach 1:
The patent removes the RNase H domain responsible for template degradation, eliminating the competitive deadnylation process. This extraction of the harmful function allows DNA synthesis to proceed without competition for the mRNA template, directly improving both productivity and product yield
3Duration of action of moving object
If RT exhibits low processivity, then dissociation from RNA template occurs frequently, but this limits DNA chain extension length
Solution Approach 1:
The patent changes the biochemical parameters of the reverse transcriptase enzyme by creating mutant versions with altered amino acid sequences. These parameter changes in the enzyme's structure and properties result in increased processivity, allowing the enzyme to remain bound to the template longer and synthesize full-length cDNA chains
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 solution results in higher DNA yield, quality, and processivity, allowing for longer DNA chain extension and improved replication fidelity, leading to higher yields of full-length cDNA with enhanced gene representation.
Implementation Method 1
Reverse transcription is a critical step in the life cycle of all RNA tumor viruses, also known as retroviruses because the retroviruses integrate their DNA into the host cell DNA by a reverse transcriptase (RT), also known as an RNA-dependent DNA polymerase, wherein the RT directs the synthesis of a complementary DNA (cDNA) from an RNA template.
Implementation Method 2
RNase H activity, on the other hand, degrades RNA from RNA-DNA hybrids, such as are formed during reverse transcription of an RNA template.
Implementation Method 3
Development of a linker-bridged gene or domain fusion reverse transcriptase enzyme with modified or deleted RNase H domains, combined with mutated RNase A, to enhance affinity for RNA-primers and RNA-DNA hybrids, thereby reducing template degradation and improving processivity and fidelity.
Data Source
AI summary
The present invention relates to combinations of a linker bridged gene or domain fusion reverse transcriptase enzyme, and more particularly, combinations of a linker bridged gene or domain fusion reverse transcriptase enzyme and their fusion construction utilizing for more efficient and quality DNA synthesis in reverse transcription. The composition of the invention includes a polymerase domain; a linker, consisting of 3-40 amino acids; and an RNase H domain, wherein the RNase H domain is either unmodified or modified with point mutations. The composition may further include another mutated RNase H, a mutated RNase A, and an additional linker which consists of 3-40 amino acids.