RNA Replicase Cycling with RdRp Binding Sites for Pure mRNA Amplification
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Solution Overview
Problem
Existing RNA replicase-mediated cycling reaction (RCR) methods require 5′-cap-capture-molecule-linked primers, which contaminate RNA products and are inefficient for mRNA vaccine production, and use 3′-CSE sequences that are too long and structural, hindering efficient transcription and enzyme availability.
Innovation Solution
Incorporate coronaviral and hepatitis C viral replicase/RNA-dependent RNA polymerase (RdRp) binding sites into the 5′- or 3′-ends of RNA templates for amplification, using RT-PCR to embed these sites in primers, allowing high-purity RNA amplification without 5′-cap-capture primers and enabling efficient transcription with helicase activity to reduce secondary structures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If 5′-cap-capture-molecule-linked primers are used in RCR, then RNA amplification can be achieved, but the RNA products are contaminated and require tedious purification that may cause RNA degradation
Solution Approach 1:
The invention extracts and removes the harmful 5′-cap-capture-molecule-linked primers from the RCR system, replacing them with simpler primers that do not contaminate the RNA products, thereby achieving high-purity RNA amplification without tedious purification steps
Solution Approach 2:
The invention uses short-lived, easily degradable primer molecules that do not require complex purification, allowing for simple removal of primers after amplification without risking RNA degradation, thus maintaining high product purity
2Productivity
If 3′-CSE sequences are used in RCR, then RNA amplification can occur, but the sequences are too long and structural, hindering efficient transcription and enzyme availability
Solution Approach 1:
The invention segments the long 3′-CSE sequence into shorter functional elements, creating a simplified binding site that retains the essential RNA replicase interaction capability while reducing overall sequence length and structural complexity for more efficient transcription
Solution Approach 2:
The invention changes the parameters of the binding site by reducing its length and simplifying its secondary structure, thereby improving accessibility for RNA replicase enzymes while maintaining amplification efficiency through optimized sequence composition
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
Achieves high-purity RNA amplification rates of 15 to over 1000 folds per cycle, facilitating the production of mRNA vaccines and RNA-based medicines for treating various diseases and generating proteins, peptides, and antibodies.
Implementation Method 1
uses RNA-dependent RNA polymerases (RdRp) to amplify single-stranded RNA sequences from RNA templates
Implementation Method 2
use reverse transcription-polymerase chain reaction (RT-PCR) to incorporate at least a coronaviral and/or HCV replicase/RdRp-binding site
Implementation Method 3
enabling efficient transcription with helicase activity to reduce secondary structures
Data Source
AI summary
This invention generally relates to a novel RNA/mRNA production and amplification method using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes as well as the associated mRNAs thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least an RdRp-binding site in the 5′- or 3′-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but also for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp), particularly coronaviral and hepatitis C viral (HCV) RdRp enzymes.