Phosphorothioate Linkers for Absolute RNA Quantification
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Solution Overview
Problem
Current RNA sequencing methods are limited in their ability to achieve absolute quantification of RNA molecules within a single sample, as they allow only relative quantification and are biased by linker ligation efficiency and encounters with modified nucleotides, leading to loss of information and sequence-dependent biases.
Innovation Solution
A novel method involving dephosphorylation, ligation with ddNTP-ended oligodeoxynucleotide linkers, reverse transcription, and subsequent ligation with hairpin/splint oligodeoxynucleotide linkers, followed by amplification and sequencing, which enables direct correlation between sequencing read counts and RNA molecule copies, minimizing biases and loss of RNA molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional RNA sequencing methods are used, then relative quantification of RNA molecules can be achieved, but absolute quantification is not possible due to linker ligation bias and modified nucleotide interference
Solution Approach 1:
The patent changes the chemical parameter of the nucleotide by using phosphorothioate-modified nucleotides instead of conventional nucleotides. This chemical modification makes the nucleotide resistant to degradation by nucleases and reduces bias during linker ligation, enabling both accurate and reliable absolute quantification of RNA molecules
Solution Approach 2:
The patent introduces phosphorothioate-modified linkers as intermediary molecules that mediate between the RNA molecule and the sequencing platform. These modified linkers serve as protective intermediaries that prevent degradation and reduce sequence-dependent biases during the sequencing process
2Ease of manufacture
If standard oligonucleotide linkers are used for RNA sequencing, then ligation can proceed, but modified nucleotides cause loss of information and sequence-dependent biases
Solution Approach 1:
The patent modifies the chemical parameter of the linker oligonucleotide by incorporating phosphorothioate modifications. This changes the chemical stability and reactivity of the linker, making it resistant to degradation and reducing sequence-dependent biases during ligation, thereby preventing information loss
Solution Approach 2:
The patent uses phosphorothioate-modified linkers as disposable intermediary molecules that are designed to be stable and resistant to degradation. These modified linkers can be used efficiently without being lost or degraded during the sequencing process
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 allows for absolute quantification of RNA molecules, providing a linear correlation between read counts and RNA copies, overcoming previous limitations and enabling accurate analysis of RNA landscapes and behaviors.
Implementation Method 1
dephosphorylating RNA in a sample, optionally using alkaline phosphatase
Implementation Method 2
ligating, to the dephosphorylated RNA, a ddNTP-ended oligodeoxynucleotide linker
Implementation Method 3
reverse transcribing the linker-ligated RNA into cDNA using a primer complementary to Linker 1 and reverse transcriptase
Implementation Method 4
employing phosphorothioates
Data Source
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AI summary
This disclosure provides methods and compositions for analyzing nucleic acids such as DNA and RNA, and including determination of absolute numbers of such nucleic acids and/or detection and localization of lesions or other modifications on such nucleic acids.