RdRp-Mediated RNA Extension for Modified-Nucleotide Sequencing
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Solution Overview
Problem
Existing RNA sequencing technologies suffer from low accuracy and yield in sequencing long RNA molecules, particularly those with modified nucleic acids, and existing methods struggle to produce long RNA molecules with modified nucleotides efficiently.
Innovation Solution
Utilizing RNA-dependent RNA polymerases, such as 3Dpol from poliovirus, to extend RNA molecules from the 3' end, incorporating modified nucleotides, and employing methods like splint ligation to synthesize long RNA molecules with modified nucleic acids, followed by nanopore sequencing to enhance accuracy and yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RNA molecules are extended using RNA-dependent RNA polymerase, then sequencing accuracy is improved by distinguishing modified nucleotides, but the complexity of the preparation method increases
Solution Approach 1:
The patent introduces RNA-dependent RNA polymerase as an intermediary enzyme to extend RNA molecules and create complementary sequences. This intermediary mechanism enables the detection of modified nucleotides by generating readable sequence information that can be compared against the original RNA, thereby improving sequencing accuracy without directly modifying the sequencing process itself
Solution Approach 2:
The patent divides the RNA molecule into segments by extending from the 3' end and creating complementary sequences. This segmentation allows for systematic analysis of different regions of the RNA molecule, enabling modified nucleotides to be identified through comparison of original and extended sequences, thus improving measurement precision
2Productivity
If splint ligation is used to synthesize long RNA molecules with modified nucleic acids, then the yield of long RNA molecules is improved, but the manufacturing complexity increases
Solution Approach 1:
The patent employs splint ligation to pre-assemble RNA fragments with modified nucleic acids into complete long RNA molecules before sequencing. This preliminary assembly action ensures that modified nucleotides are correctly positioned and integrated in the final RNA product, improving the yield of functional long RNA molecules with desired modifications
Solution Approach 2:
The patent uses splint molecules as intermediaries to facilitate the ligation of RNA fragments. These splint molecules provide a template that guides the correct alignment and joining of RNA segments containing modified nucleic acids, thereby improving assembly efficiency and yield while managing the complexity of synthesizing long modified RNA molecules
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 method improves RNA sequencing accuracy by distinguishing modified nucleotides and achieves high yield of long RNA molecules with modified nucleotides, enhancing sequencing precision and efficiency.
Implementation Method 1
contacting the RNA molecule with an RNA-dependent RNA polymerase (RdRp) in the composition, wherein the RdRp extends the 3' end of the RNA molecule using the RNA molecule as a template
Implementation Method 2
sequencing the extended RNA molecule in the first RNA composition, wherein the sequencing comprises nanopore sequencing
Data Source
AI summary
Disclosed herein are methods of preparing an RNA sample for sequencing. In certain embodiments, the method includes contacting an RNA molecule in the sample with an RNA-dependent RNA polymerase (RdRp) such that the RdRp extends the RNA molecule from the 3′ end of the RNA molecule using the RNA molecule as a template. Also disclosed herein are kits for preparing an RNA sample for sequencing according to certain methods, as well as methods of sequencing RNA molecules using the prepared sample. Also disclosed herein are methods of preparing an RNA molecule with a modified base. In certain embodiments, the method includes ligating a left-arm RNA segment, a middle RNA segment including the modified base, and a right-arm RNA segment in the presence of a DNA splint and DNA disruptors.