Poly(A)-ClickSeq Click Ligation for Unbiased 3′-End RNA Sequencing
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Solution Overview
Problem
Existing methods for sequencing poly(A) tail positions in RNA are complex, require high-depth sequencing, and often introduce sample bias and reduce throughput, limiting their application to experienced laboratories.
Innovation Solution
A method involving reverse transcription with 3'-azido-nucleotides to terminate cDNA fragments upstream of the poly(A) tail, followed by click-chemistry ligation with functionalized adaptors, enabling enrichment of poly(A) site junctions without fragmentation or enzymatic ligation, and subsequent amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing methods for sequencing poly(A) tail positions are used, then sequencing coverage is achieved, but the process complexity increases and throughput decreases
Solution Approach 1:
The patent extracts only the essential function of poly(A) tail detection by using oligo(dT) priming to specifically capture poly(A)-containing RNAs, eliminating the need for complex enrichment procedures. This extraction approach isolates the critical detection step while removing unnecessary complexity from the overall sequencing workflow.
Solution Approach 2:
The patent employs universal oligo(dT) primers that can bind to poly(A) tails across all eukaryotic mRNAs, creating a multi-functional approach that works for diverse RNA samples without requiring sample-specific optimization. This universal priming strategy simplifies the process while maintaining broad applicability and sequencing coverage.
2Measurement precision
If high-depth sequencing is performed to detect poly(A) sites, then detection sensitivity improves, but sequencing cost and time increase
Solution Approach 1:
The patent performs preliminary enrichment of poly(A) tail-containing fragments through oligo(dT) priming and selective reverse transcription before sequencing. This preliminary action concentrates the target sequences of interest, enabling detection sensitivity to be achieved with lower sequencing depth, thereby reducing both time and cost while maintaining measurement precision.
3Adaptability or versatility
If RNA fragmentation and enzymatic ligation are used in sequencing preparation, then library complexity increases, but sample bias is introduced
Solution Approach 1:
The patent utilizes the inherent poly(A) tail structure of eukaryotic mRNAs as a natural binding site for oligo(dT) primers, eliminating the need for external fragmentation and ligation enzymes. This self-service approach leverages the RNA's own features to achieve library preparation, maintaining adaptability while avoiding the introduction of enzymatic bias.
4Measurement precision
If complex enrichment procedures are applied for poly(A) sequencing, then sequencing accuracy improves, but ease of operation decreases
Solution Approach 1:
The patent replaces complex mechanical enrichment procedures with a biochemical approach using oligo(dT) priming during reverse transcription. This substitution achieves sequencing accuracy through specific base-pairing interactions rather than mechanical manipulation, significantly improving ease of operation while maintaining measurement precision.
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
Enables high-quality, unbiased sequencing libraries with reduced complexity and cost, allowing sensitive detection of poly(A) sites and APA regulation in various samples, including minimal RNA amounts and diverse applications.
Implementation Method 1
performing reverse transcription of the RNA with a reverse transcriptase primed with the adaptor sequence-oligo-dT to form terminated cDNA fragments
Implementation Method 2
combining the RNA with three terminating nucleotides of modified-deoxyGTP, modified-deoxyCTP and modified-deoxyATP
Implementation Method 3
chemically ligating a functionalized 5′ adaptor to the terminated cDNA
Implementation Method 4
The azido-group at the 3′ end of the cDNA fragment can then be used to click-ligate the cDNA to a functionalized adaptor
Data Source
AI summary
The present invention includes a method and kit for cDNA synthesis of a 3′UTR/poly(A) tail junction of cellular RNA comprising: obtaining RNA comprising a 3′UTR/poly(A) junction and a poly(a) tail; combining the RNA with three terminating nucleotides of modified-deoxyGTP, modified-deoxyCTP and modified-deoxyATP, dNTPs, and adaptor sequence-oligo-dT; performing reverse transcription of the RNA with a reverse transcriptase primed with the adaptor sequence-oligo-dT to form terminated cDNA fragments that are stochastically terminated upstream of the 3′UTR/poly(A) junction, but not within the poly(A) tail; isolating the terminated cDNA fragments; chemically ligating a functionalized 5′ adaptor to the terminated cDNA; and amplifying the chemically-ligated cDNA into an amplification product, wherein the cDNA is enriched for sequences at the 3′UTR/poly(A) tail junction without fragmentation or enzymatic ligation.