Single-Subunit RNA Polymerase for Thermostable RNA Synthesis
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Solution Overview
Problem
There is a need for alternative RNA polymerases that are more thermostable and have higher tolerance to non-canonical ribonucleoside triphosphates for biotechnological applications, particularly in the synthesis of RNA, including mRNA therapeutics and vaccines.
Innovation Solution
A novel single-subunit RNA polymerase (ssRNAP) with an amino acid sequence similar to SEQ ID NO: 1 or having at least 75% sequence identity, exhibiting enhanced thermostability and salt tolerance, and capable of efficient RNA synthesis at various temperatures, including 45°C and 55°C, with higher activity compared to T7 RNA polymerase.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional RNA polymerases (e.g., T7 RNA polymerase) are used for RNA synthesis, then the transcription efficiency and accuracy are good, but the thermostability is insufficient and the tolerance for non-canonical ribonucleoside triphosphates is limited
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the RNA polymerase to achieve enhanced thermostability. The novel ssRNAP from phage EFN4HR8C exhibits optimal activity at 37°C but maintains significant activity (52%) at 55°C, demonstrating improved thermal stability through sequence optimization while preserving catalytic function
2Adaptability or versatility
If conventional RNA polymerases are used, then the structural simplicity and promoter specificity are maintained, but the tolerance for non-canonical ribonucleoside triphosphates is insufficient for mRNA therapeutics production
Solution Approach 1:
The novel ssRNAP demonstrates universality by accommodating both canonical and non-canonical ribonucleoside triphosphates. The enzyme can incorporate modified nucleotides like pseudouridine and N1-methylpseudouridine into RNA transcripts, enabling its use in mRNA therapeutics production while maintaining transcription accuracy through its evolved active site architecture
3Duration of action of stationary object
If existing RNA polymerases are used for in vitro transcription, then the cloning and expression are efficient, but the stability under elevated temperature conditions is inadequate
Solution Approach 1:
The novel ssRNAP incorporates structural features that provide beforehand cushioning against thermal denaturation. The enzyme maintains stability during storage and exhibits resistance to thermal inactivation, allowing prolonged use at elevated temperatures without loss of activity, thus cushioning against the typical degradation issues of conventional polymerases
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 novel ssRNAP maintains high activity at elevated temperatures and tolerates non-canonical ribonucleoside triphosphates, enabling efficient RNA production, including mRNA therapeutics and vaccines, with improved stability and efficiency over existing RNA polymerases.
Implementation Method 1
a DNA-directed (DNA-dependent) RNA polymerase, i.e. a DNA-directed nucleoside-triphosphate:RNA nucleotidyltransferase, catalyzing the DNA-template-directed extension of the 3'-end of an RNA strand by one nucleotide at a time
Data Source
AI summary
A novel DNA-directed single-subunit RNA polymerase, ssRNAP, the ssRNAP having advantageous features compared to the well-known T7 RNAP.