Circular RNA Ligation Using Split RNA Elements Without Splints
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Solution Overview
Problem
Current methods for producing circular RNAs (circRNAs) with coding ability are inefficient and introduce insertions or deletions, often requiring splint oligonucleotides, leading to inaccurate ligation and polymeric by-products.
Innovation Solution
A method involving linear RNA precursors with engineered split portions forming a stable double-stranded region and a nick, ligated by RNA ligase to produce circRNAs without splint oligonucleotides, ensuring high yield and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If T4 RNA ligase 1-mediated ligation is used to cyclize RNA, then circular RNAs can be produced, but insertions and deletions are introduced during the ligation process resulting in inaccurate ligation
Solution Approach 1:
A splint oligonucleotide is introduced as an intermediary molecule to facilitate accurate ligation. The splint hybridizes to complementary sequences on the linear RNA ends, bringing them into proper alignment for ligation by T4 RNA ligase 1, thereby preventing insertions and deletions while enabling circular RNA formation
Solution Approach 2:
The splint oligonucleotide serves as a template or copy that defines the correct spatial arrangement for ligation. By providing a predefined sequence that complements both ends of the linear RNA, it ensures that the ligation occurs at the precise intended location without introducing errors
2Productivity
If splint oligonucleotide is used to bring the two ends of linear RNA together for ligation, then efficient ligation can be achieved, but polymeric by-products form and the splint must be removed
Solution Approach 1:
The harmful polymeric by-products and the splint oligonucleotide itself are extracted or removed from the final circular RNA product through purification steps, yielding clean circular RNA without contaminating sequences
Solution Approach 2:
The splint oligonucleotide, after serving its purpose of facilitating ligation, is discarded or removed from the reaction mixture. The circular RNA product is recovered in a purified state, free from the splint and polymeric by-products
3Ease of manufacture
If splint oligonucleotide is required for ligation, then the process can proceed, but additional sequences are introduced that may be immunogenic or reduce cargo size
Solution Approach 1:
The splint oligonucleotide is designed as a temporary, disposable element that is present only during the ligation reaction. It is completely removed afterward, leaving no permanent sequence in the final circular RNA product, thus avoiding immunogenicity and cargo size issues
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 method achieves high-efficiency and accurate production of circRNAs, promoting translation of coding sequences while avoiding additional sequences that could be immunogenic or reduce cargo size.
Implementation Method 1
the first portion of the RNA element and the second portion of the RNA element associate with each other to form a double-stranded region
Implementation Method 2
the nick can be ligated by a RNA ligase (e.g., T4 RNA ligase 1 or T4 RNA ligase 2)
Data Source
Figure 1
Figure 2A
Figure 2B~2C
AI summary
Provided are linear RNA precursors and constructs for preparing circular RNAs (circRNAs) comprising from the 5' end to the 3' end: (a) a first portion of an RNA element (such as an IRES), (b) an effector RNA sequence, and (c) a second portion of the RNA element, wherein the first portion of the RNA element and the second portion of the RNA element associate with each other to form a double-stranded region of at least 4 basepairs (bp) long, wherein the 5' end of the first portion of the RNA element and the 3' end of the second portion of the RNA element form a nick in the double-stranded region, and wherein the nick can be ligated by a RNA ligase. Also provided are methods of preparing circRNAs, and circRNAs prepared thereof.