Codon-Optimized Nucleotide Sequence Screening via Plasmid Assembly
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Solution Overview
Problem
Current methods for screening codon-optimized protein-coding nucleotide sequences are resource intensive and difficult to perform at scale, hindering the identification of optimized sequences suitable for efficient commercial production of full-length mRNA transcripts at an early development stage.
Innovation Solution
The method involves dividing nucleotide sequences generated by a codon optimization algorithm into DNA fragments with overlapping homologous ends, which are then efficiently assembled using advanced molecular biology techniques like Gibson assembly. This assembly process creates plasmids with the nucleotide sequences flanked by 5′ and 3′ untranslated regions and linked to an RNA polymerase promoter, enabling rapid in vitro transcription of full-length mRNA transcripts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current screening methods are used for codon-optimized nucleotide sequences, then sequence optimization can be performed, but the process becomes resource intensive and difficult to perform at scale
Solution Approach 1:
The patent divides the nucleotide sequence into multiple DNA fragments with overlapping homologous ends, which are then assembled into plasmids. This segmentation allows parallel processing of multiple sequences simultaneously, enabling high-throughput screening while reducing the complexity of individual processing steps.
Solution Approach 2:
The patent introduces plasmids as intermediary carriers that contain the codon-optimized nucleotide sequences flanked by 5' and 3' untranslated regions and linked to an RNA polymerase promoter. This intermediary system enables standardized, scalable screening of multiple sequences in a uniform platform, dramatically improving throughput.
2Reliability
If codon optimization is performed to increase protein expression, then protein expression levels improve, but cryptic transcription termination sites or sequence motifs may be introduced that interfere with efficient transcription
Solution Approach 1:
The patent incorporates 5' and 3' untranslated regions and RNA polymerase promoters in the plasmid construct before transcription occurs. This preliminary setup ensures that transcription initiation and termination are properly controlled, preventing cryptic termination sites from interfering with the expression of codon-optimized sequences.
Solution Approach 2:
The patent uses the plasmid structure with flanking untranslated regions and promoters to convert potential harmful transcription termination effects into beneficial controlled transcription. The designed plasmid structure ensures that only full-length mRNA transcripts are produced, transforming potential transcriptional interference into reliable expression control.
3Reliability
If multiple candidate optimized nucleotide sequences are generated and screened, then the likelihood of finding high-expression sequences increases, but the time and resources required for screening increase
Solution Approach 1:
The patent merges multiple candidate nucleotide sequences into a single plasmid-based screening platform. By incorporating multiple sequences flanked by universal untranslated regions and promoters, the system enables parallel evaluation of many candidates simultaneously, reducing total screening time while maintaining high reliability through comprehensive sequence testing.
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
This approach allows for the rapid screening of large numbers of optimized nucleotide sequences, enabling the production of full-length mRNA transcripts with high in vivo potency within a couple of weeks, thus facilitating early-stage commercial development.
Implementation Method 1
assembled via homologous ends into plasmids that comprise the nucleotide sequences of interest
Implementation Method 2
operationally linked to an RNA polymerase promoter... transcribe the nucleotide sequence into an mRNA transcript
Data Source
AI summary
The present invention relates to methods for screening protein-coding nucleotide sequences generated by a codon optimization algorithm to identify those sequences that generate a full-length mRNA transcript, and optionally, high protein expression. In particular, the present invention relates to screening methods wherein a plurality of protein-coding nucleotide sequences is provided as two or more DNA fragments which are assembled via homologous ends into plasmids that comprise the nucleotide sequences of interest flanked by a 5′ untranslated region (5′ UTR) and a 3′ untranslated region (3′ UTR) and operationally linked to an RNA polymerase promoter.