RtcB Ligase RNA Ligation for Long Short Molecule Differentiation
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Solution Overview
Problem
Current methods for processing RNA samples are inefficient in differentiating and analyzing long and short RNA molecules, as they often result in non-specific fragmentation and lack effective techniques for separating and sequencing these diverse RNA types.
Innovation Solution
A method involving fragmentation of long RNA molecules to produce 5'-OH and 2',3'-cyclic phosphate termini, followed by ligation with adaptors using RtcB ligase, and subsequent separation and sequencing of both long and short RNA fragments, utilizing specific adaptors and enzymes to create a ligated RNA sample suitable for next-generation sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional RNA processing methods are used, then RNA samples can be processed, but long and short RNA molecules cannot be efficiently differentiated and analyzed
Solution Approach 1:
The method segments the RNA analysis process into distinct pathways: long RNA molecules are fragmented to generate 5'-OH and 2',3'-cyclic phosphate termini, while short RNA molecules remain intact. This segmentation allows differential processing and analysis of the two RNA types, resolving the contradiction between differentiation precision and analysis efficiency.
Solution Approach 2:
RtcB ligase serves as an intermediary enzyme that specifically recognizes and ligates the 2',3'-cyclic phosphate termini generated from long RNA fragmentation. This intermediary mechanism enables selective identification and analysis of long RNA-derived fragments amidst the total RNA population, improving differentiation precision without compromising overall analysis productivity.
2Ease of manufacture
If fragmentation is applied to RNA samples, then long RNA molecules can be processed, but non-specific fragmentation occurs affecting short RNAs
Solution Approach 1:
The method changes the fragmentation parameters by using RtcB ligase activity as a selective marker. Instead of relying solely on physical fragmentation specificity, the approach uses enzymatic recognition of specific chemical termini (2',3'-cyclic phosphate) generated during fragmentation. This parameter change allows robust processing of long RNA while maintaining reliability by using enzymatic specificity rather than purely physical fragmentation control.
3Productivity
If current sequencing preparation methods are used, then RNA can be sequenced, but effective separation and analysis of diverse RNA types is lacking
Solution Approach 1:
The method performs preliminary differential processing before sequencing: long RNA molecules are fragmented and ligated with adaptors via RtcB, while short RNA molecules are protected from fragmentation or processed differently. This preliminary action preserves RNA profile information by maintaining distinct molecular signatures that can be recovered during sequencing analysis, preventing information loss while enabling comprehensive sequencing of diverse RNA types.
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 enables efficient separation and sequencing of long and short RNA molecules, allowing for detailed analysis of RNA profiles and transcriptome investigation, enhancing diagnostic and research capabilities.
Implementation Method 1
contacting said fragmented RNA sample with a first adaptor in the presence of a RtcB ligase, thereby producing a ligated RNA sample comprising adaptor-ligated fragments of long RNA
Implementation Method 2
contacting the initial RNA sample with a divalent cation at a temperature of at least 50° C.
Implementation Method 3
contacting an initial RNA sample comprising small RNA molecules having a 5′-phosphate and a 3′-OH with an enzyme, e.g., a phosphatase, that removes the 5′-phosphate from the short RNA molecules
Data Source
AI summary
A method of processing an RNA sample is provided. In certain embodiments, the method may comprise: a) obtaining a fragmented RNA sample comprising: i. RNA fragments of long RNA molecules; and ii. unfragmented short RNA; and b) contacting said fragmented RNA sample with a first adaptor in the presence of a RtcB ligase, thereby producing a ligated RNA sample comprising adaptor-ligated fragments of long RNA. A kit for performing the method is also provided.