Self-Amplifying RNA Platform via RdRP-Binding Site Design
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Solution Overview
Problem
Existing methods for amplifying RNA sequences, such as PCR, require DNA templates and primers that can cause contamination and are not compatible with certain viral RNA sequences, limiting their applicability for developing RNA-based vaccines and medicines.
Innovation Solution
A novel self-amplifying RNA/mRNA (samRNA) composition incorporating specific replicase/RNA-dependent RNA polymerase (RdRP)-binding sites at the 5′-end or 3′-end of RNA sequences, allowing amplification through Replicase- and/or RNA-dependent RNA polymerase-mediated Cycling Reaction (RCR) without DNA templates or primers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If PCR method is used to amplify RNA sequences, then amplification can be achieved, but DNA templates and primers cause contamination and are not compatible with certain viral RNA sequences
Solution Approach 1:
The invention extracts and eliminates the DNA template and primer components from the amplification system, replacing them with an all-RNA system using RdRP enzymes. This removal of DNA elements directly addresses the contamination problem while maintaining amplification capability for viral RNA sequences.
Solution Approach 2:
The invention introduces RNA-dependent RNA polymerase (RdRP) as an intermediary enzyme that enables RNA amplification without requiring DNA templates. The RdRP mediates the replication of viral RNA sequences directly, solving both the contamination issue and the incompatibility with certain viral RNA structures.
2Productivity
If DNA templates and primers are used in PCR, then amplification is possible, but the process becomes complex and requires multiple components
Solution Approach 1:
The invention removes unnecessary DNA templates and complex primer structures from the system, retaining only the essential RNA components and RdRP enzyme needed for amplification, thereby reducing overall system complexity.
Solution Approach 2:
The viral RNA sequences themselves contain or can be engineered to contain internal RdRP binding sites that serve as self-priming elements, eliminating the need for externally added primers and simplifying the amplification process.
3Quantity of substance
If conventional amplification methods are used, then RNA can be amplified, but high-yield and high-purity amplification is difficult to achieve
Solution Approach 1:
The invention designs specific RdRP binding sites with optimized local sequences that enhance the efficiency and specificity of RdRP recognition and binding, leading to improved amplification yield and purity through localized sequence optimization rather than global changes.
Solution Approach 2:
The invention optimizes parameters such as RdRP binding site sequence composition, length, and structural characteristics to maximize amplification efficiency and product purity, achieving high-yield and high-purity RNA amplification through parameter tuning.
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
Enables high-yield, high-purity amplification of RNA sequences with flexible strand configurations, suitable for generating vaccines and medicines, and can be used in various RNA-based applications, including anti-viral and anti-cancer therapies.
Implementation Method 1
uses viral replicases and/or RNA-dependent RNA polymerases (RdRP) to amplify desired single-stranded and/or double-stranded RNA/mRNA sequences from pre-designed samRNA platform templates
Implementation Method 2
incorporating at least a specific coronaviral (e.g. COVID-19-associated viruses) and/or hepatitis C viral (HCV) replicase/RdRP-binding (recognition) site into the 5'-end or 3'-end region, or both, of a desired RNA/mRNA sequence
Data Source
AI summary
This invention relates to a novel composition of RNA/mRNA medicines and/or vaccines produced by using Replicase/RNA-dependent RNA polymerase (RdRP)-mediated RNA Cycling Reaction (RCR). This RCR-amplifiable RNA/mRNA composition comprises at least a replicase/RdRP-binding site (RdRP-BS) in the 5′-end or 3′-end, or both, of a desired RNA sequence of interest, to form a self-amplifying RNA/mRNA (samRNA) platform. The samRNA platform so obtained is useful for designing and developing a variety of self-amplifying RNA/mRNA (samRNA) constructs, of which the desired RNA sequences may include, but not limited to, antisense oligonucleotide RNA (aRNA; ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA)/miRNA precursor (pre-miRNA), long non-coding RNA (lncRNA), and/or messenger RNA (mRNA), or a combination thereof. The present RdRP-BS designs in said samRNA are derived or modified from the identified RdRP-BS motifs of coronavirus (e.g. SARS-CoV-2-associated viruses) and/or hepatitis C virus (HCV) in either single-stranded or double-stranded conformation, or a combination thereof.