Triazole Linkage Nucleic Acid Processing for RNA Sequencing Bias
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Solution Overview
Problem
Current RNA sequencing methods, particularly those involving small RNAs, face significant bias due to ligation-mediated issues, where secondary structures of target RNAs in the ligation buffer lead to uneven adaptor ligation efficiency, affecting the reliability of miRNA expression level measurements.
Innovation Solution
The method employs chemical ligation using triazole linkages formed through Cu I-catalysed Azide-Alkyne Cycloaddition or Strain-Promoted Alkyne Azide Cycloaddition reactions, allowing reverse transcriptase to read through bulky triazole linkages, thereby bypassing the need for enzymatic ligation and reducing sequencing bias.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If enzymatic ligation (T4 RNA ligase) is used to attach adaptors to RNA fragments, then adaptor ligation can be achieved, but sequencing bias occurs due to secondary structures of target RNAs affecting ligation efficiency
Solution Approach 1:
The patent replaces the enzymatic ligation system (T4 RNA ligase) with a chemical ligation system using triazole linkages formed through Cu(I)-catalyzed azide-alkyne cycloaddition. This substitution eliminates the biological enzyme's sensitivity to RNA secondary structures while maintaining effective adaptor attachment, thereby resolving the sequencing bias problem without sacrificing ligation efficiency
Solution Approach 2:
The patent changes the chemical parameters of the ligation reaction by using click chemistry with triazole linkages instead of enzymatic reactions. This parameter change allows the ligation to proceed independently of the RNA's secondary structure, as the chemical reaction conditions (pH, temperature, catalyst) are not affected by the RNA folding state, thus eliminating the source of sequencing bias
2Manufacturing precision
If gel purification steps are included to separate 3'-adaptor and 5'-adaptor ligation reactions, then adaptor ligation accuracy is improved, but procedure complexity and time increase
Solution Approach 1:
The patent extracts and removes the gel purification step from the sequencing library preparation workflow. By using chemically modified adaptors with triazole linkages that can be specifically targeted and removed or retained based on their chemical properties, the method eliminates the need for gel-based separation, thereby simplifying the procedure while maintaining adaptor ligation accuracy
Solution Approach 2:
The patent introduces chemically modified adaptors containing triazole linkages as intermediaries that facilitate selective purification or removal. These modified adaptors serve as mediators that can be specifically targeted by chemical reactions or binding agents, allowing for clean separation without gel electrophoresis and simplifying the overall workflow
3Ease of manufacture
If poly-A tailing is used for 3'-handle installation, then 3'-adaptor attachment is achieved, but poly-A signal contamination and procedure complexity increase
Solution Approach 1:
The patent replaces the poly-A tailing enzymatic process with direct chemical ligation using triazole-linked adaptors. This substitution eliminates the generation of poly-A signals that contaminate sequencing data, while still achieving effective 3'-handle installation for library preparation
Solution Approach 2:
The patent converts the potential harm of poly-A tailing (signal contamination) into a benefit by using chemically modified adaptors with triazole linkages. These modified adaptors provide unique chemical handles that can be specifically recognized and processed, turning the ligation step into a precise, controllable reaction that avoids the unwanted poly-A signal generation while maintaining efficient adaptor attachment
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 enables more reliable RNA sequencing by minimizing ligation-mediated bias, allowing for larger-scale chemical ligation compatible with various pH and salt conditions, and eliminating the need for gel purification steps, thus improving the accuracy of small RNA profiling.
Implementation Method 1
processing the adapted nucleic acid fragment by transcribing at least a portion of the adapted nucleic acid fragment with reverse transcriptase, wherein said portion comprises the triazole linkage
Implementation Method 2
such triazole linkages are formed through the process of chemical ligation methods, such as a Cu I-catalysed Azide-Alkyne Cycloaddition
Data Source
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AI summary
The invention relates to a method of nucleic acid processing comprising: providing an adapted nucleic acid fragment having a triazole linkage therein; and transcribing the adapted nucleic acid fragment with reverse transcriptase. The invention further relates to kits and uses associated with the method.