Splint-Mediated RNA Ligation for Full-Length gRNA Synthesis
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Solution Overview
Problem
Current methods for synthesizing guide RNAs (gRNAs) face challenges in achieving full-length products due to limitations in chemical synthesis, particularly for lengths around 100 nucleotides, where coupling efficiency and purification of complete RNA molecules from truncation products are inefficient using standard methods.
Innovation Solution
The use of splint-mediated ligation of RNA fragments, where RNA fragments with specific terminal regions and splint oligonucleotides are hybridized and ligated using a ligase to form full-length guide RNAs, improving yield and reducing truncation products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If phosphoramidite chemistry is used to synthesize gRNAs of 100 nucleotides, then chemical synthesis can be performed, but the yield of full-length product is only 30-40% due to coupling efficiency limitations
Solution Approach 1:
The gRNA is divided into multiple smaller fragments (e.g., 3-5 fragments of 20-40 nucleotides each) that are synthesized separately using phosphoramidite chemistry with high coupling efficiency, then ligated together to form the complete 100-nucleotide gRNA. This segmentation allows each fragment to be synthesized with near-perfect yield while the ligation step efficiently assembles them.
Solution Approach 2:
The gRNA is pre-divided into optimized fragments with predetermined sequences and structures before synthesis. Each fragment is designed to have ideal length for high coupling efficiency and specific structural features (such as hairpin structures or overhangs) that facilitate efficient ligation, thereby preparing the system in advance for maximum productivity.
2Ease of manufacture
If standard purification methods are used for gRNAs of 100 nucleotides, then purification can be performed, but complete isolation of full-length product from truncation products is not achievable
Solution Approach 1:
By synthesizing the gRNA as multiple separate fragments and ligating them, the purification process benefits from the fact that unligated fragments and partial products have different sizes and properties than the complete ligation product, enabling much cleaner separation and isolation of the full-length gRNA using standard methods like gel electrophoresis or HPLC.
Solution Approach 2:
The ligation junctions between fragments create unique local structural features (such as phosphodiester bonds at specific positions or adjacent modified nucleotides) that differentiate the full-length product from truncation products, allowing purification methods to target these specific local qualities for high-purity isolation.
3Reliability
If chemical synthesis is used for gRNAs, then chemical modifications can be incorporated to increase stability and reduce immunogenicity, but truncation products are formed that reduce effectiveness
Solution Approach 1:
Chemical modifications (such as 2'-O-methyl, phosphorothioate, or locked nucleic acid modifications) are incorporated into each fragment during synthesis, ensuring that every nucleotide in the final gRNA receives the desired modification. The segmentation approach allows modifications to be applied systematically to each fragment, guaranteeing complete coverage in the final ligation product while maintaining high yield.
Solution Approach 2:
The synthesis approach changes from single-step chemical synthesis to multi-step ligation of modified fragments, allowing optimization of modification density and type in different regions of the gRNA. This parameter change enables tailored modification patterns that maximize stability and minimize immunogenicity while ensuring complete ligation to form functional full-length product.
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 method enhances the synthesis of full-length gRNAs by increasing the yield of complete products and decreasing truncation products, allowing for the production of both unmodified and modified gRNAs with improved chemical stability and reduced immunogenicity.
Implementation Method 1
hybridizing the first RNA fragment, the second RNA fragment, and the splint oligonucleotide together to form a complex
Implementation Method 2
ligating the first and second RNA fragments using a ligase at a ligation site present within the complex, thereby synthesizing an mlRNA
Data Source
AI summary
The present disclosure relates to methods of synthesizing moderate length RNAs by splint-mediated ligation of RNA fragments.