Optimizing circRNA Expression Frame Lengths for Circularization
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Solution Overview
Problem
Current methods for overexpressing circular ribonucleic acid (circRNA) face challenges in optimizing the length of the expression frame to balance the insertion of exogenous sequences while ensuring accurate back-splicing and efficient circularization, leading to complexity in splicing by-products.
Innovation Solution
A circRNA expression frame with a nucleotide sequence of 92 bp upstream and 75 bp downstream, including an EcoR I restriction site, is designed to facilitate efficient circularization and insertion of target sequences, using PCR amplification and recombination to construct a high-efficiency eukaryotic expression vector.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a longer expression frame sequence is used, then the circularization efficiency is improved, but the length of foreign gene sequence that can be inserted is reduced
Solution Approach 1:
The expression frame is divided into three functional segments: upstream frame sequence (92 bp), restriction site (6 bp), and downstream frame sequence (75 bp). This segmentation allows optimization of circularization efficiency while preserving space for foreign gene insertion, as each segment performs a specific function in the back-splicing process.
Solution Approach 2:
The patent optimizes the lengths of upstream and downstream flanking sequences to 92 bp and 75 bp respectively, representing parameter changes that balance circularization efficiency with insert capacity. These specific length parameters were determined to achieve high back-splicing efficiency while accommodating substantial foreign sequences.
2Length of moving object
If a shorter expression frame sequence is used, then the insertable foreign gene length is increased, but the circularization efficiency is reduced
Solution Approach 1:
The patent identifies optimal parameter values for the expression frame components: upstream frame sequence of 92 bp and downstream frame sequence of 75 bp. These parameter changes represent the minimum lengths required to maintain high circularization efficiency while maximizing foreign gene insertion capacity.
3Measurement precision
If longer flanking sequences are used for circularization, then the back-splicing accuracy is improved, but the splicing by-products complexity increases
Solution Approach 1:
The patent optimizes flanking sequence lengths to 92 bp upstream and 75 bp downstream, representing parameter changes that achieve high back-splicing accuracy while minimizing splicing by-products. These specific lengths provide sufficient complementarity for accurate pairing without excessive sequence complexity that would generate unwanted by-products.
Data Source
AI summary
Provided is a high-efficiency expression vector of a circular ribonucleic acid (circRNA). The high-efficiency expression vector is an expression framework with a total length of only 173 bp and having a nucleotide sequence shown in SEQ ID NO: 1, and includes an upstream frame sequence of 92 bp, a downstream frame sequence of 75 bp, and a restriction site EcoR I located between the upstream and downstream frame sequences for inserting a gene to be circularized. In the present disclosure, the high-efficiency expression vector of a circRNA can enable an exogenously-inserted sequence to efficiently express the circRNA, thus providing an efficient expression tool for obtaining the circRNA. On the premise of ensuring efficient circularization of the circRNA, lengths of upstream and downstream flanking sequences required for the circularization have been highly optimized to further reduce the complexity of splicing by-products caused by the flanking sequences.