Codon-Optimized Recombinant Vector for RdRp Recognition and RNA Amplification
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Solution Overview
Problem
Existing RNA-dependent RNA cycling reactions (RCR) face challenges in achieving satisfactory amplification results due to the limitations of viral replicases in recognizing RdRp-binding sites, necessitating an improved replicase for efficient in vitro RNA amplification.
Innovation Solution
A recombinant vector optimized with human codons expresses a novel polydeoxyribonucleotide (PDRN) encoding a replicase, capable of recognizing RNA templates to initiate transcription, facilitating RNA amplification through RNA-dependent RNA cycling reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If viral replicases are used in RNA-dependent RNA cycling reactions, then the reaction can proceed with existing enzymatic tools, but the amplification results are unsatisfactory due to poor recognition of RdRp-binding sites
Solution Approach 1:
The patent modifies the amino acid sequence of the replicase enzyme through site-directed mutagenesis to optimize its binding affinity and specificity for the RdRp-binding site. By changing specific amino acid residues in the replicase protein structure, the enzyme's ability to recognize and bind to the RNA template is enhanced, thereby improving amplification efficiency while maintaining enzymatic function.
Solution Approach 2:
The patent creates a recombinant replicase gene that encodes a modified replicase protein with improved binding properties. This recombinant gene serves as a copy of the original viral replicase gene but with optimized sequences that enhance the enzyme's ability to recognize RdRp-binding sites, allowing the system to overcome the limitations of wild-type viral replicases.
2Productivity
If a recombinant vector with optimized polydeoxyribonucleotide is used to express replicase, then the replicase expression is enhanced with human codons, but the vector complexity increases
Solution Approach 1:
The patent applies codon optimization by replacing viral or bacterial codons with human-preferred codons in the replicase gene sequence. This parameter change in the nucleotide sequence increases the efficiency of translational machinery in human cells, leading to higher replicase expression levels. The optimization maintains the same amino acid sequence while improving gene expression through codon usage bias adjustment.
Solution Approach 2:
The recombinant vector serves as an intermediary carrier that introduces the optimized replicase gene into human cells. The vector includes necessary regulatory elements such as promoters and origin of replication that mediate gene expression and maintenance, enabling high-level replicase production without requiring direct manipulation of the human genome.
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 recombinant vector enables efficient amplification of RNA products, including coding and non-coding RNAs, with high specificity and yield, suitable for producing amplified RNA products such as cancer, bacterial, viral, and fungal antigens, and parasitic antigens.
Implementation Method 1
the polydeoxyribonucleotide of SEQ ID NOs: 1, 2 or 3 is independently transcribed to a polyribonucleotide sequence of SEQ ID NOs: 4, 5, or 6
Implementation Method 2
The RCR process relies mainly on the participation of RNA-dependent RNA polymerase (RdRp) for replication
Data Source
AI summary
Disclosed herein is a recombinant vector for expressing a replicase comprising a novel polydeoxyribonucleotide. The thus-expressed replicase can be used in in vitro RNA amplification via an RNA-dependent RNA cycling reaction (RCR). Also disclosed herein is a method for producing an amplified RNA product in an RNA cycling reaction (RCR) via use of the present replicase encoded by the present polydeoxyribonucleotide.