Synthetic gRNA Ligation via Stem-Loop Structures
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Solution Overview
Problem
The synthesis of guide RNA (gRNA) molecules is hindered by challenges such as the limitations of phosphoramidite chemistry, which results in low coupling efficiency, incomplete purification, and the presence of side products that reduce the chemical stability and increase off-target editing.
Innovation Solution
A ligation-based approach is employed using enzyme-mediated ligation of partially complementary synthetic RNAs to form stem structures, increasing the purity, yield, and integrity of gRNA, thereby enhancing editing efficiencies and reducing off-target editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If phosphoramidite chemistry is used to synthesize gRNA, then the gRNA can be produced, but the coupling efficiency is low and side products are formed
Solution Approach 1:
The gRNA synthesis is divided into two stages: first synthesizing shorter RNA fragments (20-50 nucleotides) that can be efficiently produced by phosphoramidite chemistry with high purity, then ligating these fragments together to form the full-length gRNA. This segmentation allows each synthesis step to operate at optimal efficiency while avoiding the accumulation of side products that would occur in a single long synthesis reaction.
Solution Approach 2:
The method performs preliminary synthesis of individual RNA fragments before final assembly. By pre-synthesizing and purifying shorter fragments separately, the protocol ensures high purity components are available before ligation, preventing side products from compromising the final product quality.
2Productivity
If standard purification methods are used for gRNA, then the full-length product can be isolated, but side products with similar sequence homology cannot be completely removed
Solution Approach 1:
By segmenting the synthesis into separate fragment production and ligation steps, the method enables purification of individual fragments before assembly. This intermediate purification stage allows standard methods to effectively remove side products from each fragment, and the ligation step joins only the purified fragments, ensuring high purity in the final product.
3Length of moving object
If longer gRNA molecules are synthesized, then the therapeutic applications are enabled, but the coupling efficiency decreases and side products increase
Solution Approach 1:
The method synthesizes longer gRNA molecules by segmenting them into shorter, efficiently synthesizable fragments (20-50 nucleotides each) that are then ligated together. This approach maintains high coupling efficiency in each synthesis step while achieving the required total length for therapeutic applications, avoiding the exponential decrease in efficiency that would occur with direct synthesis of the full-length molecule.
Solution Approach 2:
The method combines multiple efficiently-synthesized fragments through enzymatic ligation to create the full-length gRNA. By merging pre-synthesized high-purity fragments, the protocol achieves both the required length and high coupling efficiency, as each fragment is produced under optimal conditions before being joined to form the complete therapeutic molecule.
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 ligation-based method significantly improves the purity, yield, and integrity of gRNA, leading to increased editing efficiencies and reduced off-target effects compared to conventional synthesis methods.
Implementation Method 1
A ligation-based approach is employed using enzyme-mediated ligation of partially complementary synthetic RNAs to form stem structures
Data Source
AI summary
The present invention provides, among other things, a method of producing a synthetic RNA using a self-templating approach. For example, in some embodiments a synthetic gRNA is produced comprising: contacting a first RNA with a second RNA, wherein the first RNA and the second RNA comprise at least five RNA nucleotides that are complementary, and wherein the contacting forms a stem structure or a stem loop structure, and ligating the first RNA and the second RNA with a ligating enzyme (i) within the stem structure, or (ii) at an end of the stem structure, thereby forming a loop at the end of the stem structure.