Engineered T7 RNA Polymerase Fusion for Capped RNA Transcripts
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Solution Overview
Problem
The lack of 5' modifications in T7 RNA polymerase (RNAP) derived transcripts limits its use in eukaryotic chassis organisms and in vitro production of functional eukaryotic mRNAs, with existing fusion enzymes showing lower capping efficiency compared to Pol II derived transcripts.
Innovation Solution
Development of an engineered enzyme comprising a T7 RNA polymerase component and a capping enzyme component separated by a linker, with specific amino acid sequences and substitutions to enhance polymerase and capping activity, and a method for selecting variants with improved activity using eukaryotic cells and directed evolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If T7 RNA polymerase is used for transcription in eukaryotic cells, then simplicity and high transcription efficiency are achieved, but the transcripts lack 5' modifications required for eukaryotic functionality
Solution Approach 1:
The patent combines T7 RNA polymerase and capping enzyme into a single fusion protein (T7 RNAP-NP868R). This merging allows the polymerase to synthesize transcripts while simultaneously adding 5' caps, resolving the contradiction by ensuring eukaryotic functionality is achieved without sacrificing transcription efficiency.
Solution Approach 2:
The capping enzyme is incorporated into the fusion protein to perform capping action immediately during transcription. This preliminary action ensures that 5' modifications are added co-transcriptionally, preventing the loss of eukaryotic functionality while maintaining high productivity.
2Reliability
If wild type fusion enzyme (NP868R fused to T7 RNAP) is used, then capping capability is achieved, but capping efficiency is lower than Pol II derived transcripts
Solution Approach 1:
The patent applies directed evolution to change the amino acid parameters of the fusion enzyme, generating variants with optimized capping efficiency. By mutating specific residues in the NP868R domain, the enzyme achieves higher catalytic efficiency while maintaining its ability to cap transcripts, thus resolving the contradiction between capability and productivity.
Solution Approach 2:
The directed evolution process uses fluorescence-based screening to provide feedback on capping efficiency. Variants with improved efficiency are selected and iteratively refined, allowing the system to self-optimize and achieve high capping efficiency while maintaining capability.
3Ease of operation
If single subunit capping enzyme (NP868R) is used instead of vaccinia capping enzyme, then implementation simplicity is improved, but capping activity may be reduced
Solution Approach 1:
The patent optimizes the NP868R enzyme through directed evolution, changing its kinetic parameters to enhance capping activity. The evolved variants maintain the simplicity of the single-subunit enzyme while achieving improved catalytic performance, thus resolving the contradiction between ease of operation and productivity.
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 engineered enzyme achieves enhanced capping and polymerase activity, enabling efficient production of capped transcripts in eukaryotic cells, particularly in Saccharomyces cerevisiae, with increased protein expression and fluorescence levels.
Implementation Method 1
T7 RNA polymerase (RNAP) based transcription
Implementation Method 2
capping enzyme derived from African Swine Fever virus (NP868R) was able to catalyze all three reactions involved in the generation capped RNA
Data Source
AI summary
Disclosed herein is an engineered fusion enzyme composed of a prokaryotic phage RNA polymerase (T7 RNAP) and a viral capping enzyme (NP868R) for the generation of RNA transcripts containing a 5′ 7-methylguanylate cap.