Simultaneous Adaptor Ligation to RNA Molecules
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Solution Overview
Problem
Current methods for detecting and quantifying RNA molecules, particularly small non-coding RNAs, are inefficient and lack specificity, often requiring sequential ligation steps and purification processes, which can lead to incomplete detection and analysis of RNA populations.
Innovation Solution
A method involving a ligation reaction composition with specific adaptors and a double-strand specific RNA ligase, where both adaptors are ligated simultaneously to the RNA molecule, followed by reverse transcription and amplification using RNA-directed DNA polymerase, allowing for the detection and quantification of RNA species through amplification and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If sequential ligation steps are used for adaptor ligation, then the ligation process can be performed with standard enzymes, but the detection completeness and analysis efficiency are reduced
Solution Approach 1:
The patent combines two separate ligation steps into a single simultaneous ligation reaction. Both the 5' adaptor and 3' adaptor are ligated to the RNA molecule in the same reaction mixture using a double-strand specific RNA ligase, eliminating the need for sequential processing and intermediate purifications.
Solution Approach 2:
The patent designs adaptors with pre-formed double-stranded regions that hybridize to the RNA molecule before ligation. This preliminary hybridization ensures proper positioning and orientation of the adaptors, enabling the ligase to efficiently complete the ligation reaction in a single step.
2Manufacturing precision
If sequential ligation with intermediate purification is performed, then each ligation step can be optimized independently, but the overall process time and complexity increase
Solution Approach 1:
The patent merges multiple process steps (first ligation, purification, second ligation) into a single combined reaction. The reaction mixture contains all necessary components for both adaptors to ligate simultaneously, eliminating intermediate purification steps and reducing overall process complexity.
Solution Approach 2:
The double-strand specific RNA ligase performs multiple functions: it ligates both the 5' adaptor and 3' adaptor to the RNA molecule in a single reaction. This multi-functional enzyme replaces what would traditionally require multiple specialized enzymatic steps.
3Ease of operation
If standard ligation methods are used, then the procedure is simpler, but the specificity for double-stranded RNA regions is reduced
Solution Approach 1:
The patent uses a double-strand specific RNA ligase as a specialized mediator that recognizes and binds only to double-stranded RNA structures. This enzyme acts as a selective gatekeeper, ensuring ligation occurs only at properly hybridized adaptor-RNA interfaces, thereby enhancing specificity while maintaining procedural simplicity.
Solution Approach 2:
The patent changes the key parameter of enzyme specificity by employing a double-strand specific RNA ligase instead of standard T4 RNA ligase. This parameter change enables the reaction to distinguish between properly hybridized double-stranded regions and single-stranded regions, improving measurement precision without complicating the overall procedure.
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 efficient and simultaneous ligation of adaptors to RNA molecules, facilitating comprehensive detection and quantification of RNA species, including small non-coding RNAs, with improved specificity and sensitivity, enabling detailed analysis of RNA populations.
Implementation Method 1
a double-strand specific RNA ligase, wherein the first adaptor and the second adaptor are ligated to the RNA molecule in the ligation reaction composition
Implementation Method 2
followed by reverse transcription and amplification using RNA-directed DNA polymerase
Implementation Method 3
amplification using RNA-directed DNA polymerase, allowing for the detection and quantification of RNA species through amplification and sequencing
Data Source
AI summary
Compositions, methods, and kits for detecting one or more species of RNA molecules are disclosed. In one embodiment, a first adaptor and a second adaptor are ligated to the RNA molecule using a polypeptide comprising double-strand specific RNA ligase activity, without an intervening purification step. The ligated product is reverse transcribed, then at least some of the ribonucleosides in the reverse transcription product are removed. Primers are added and amplified products are generated. In certain embodiments, the sequence of at least part of at least one species of amplified product is determined and at least part of the corresponding RNA molecule is determined. In some embodiments, at least some of the amplified product species are detected, directly or indirectly, allowing the presence and/or quantity of the RNA molecule of interest to be determined.