3' Blocked Nucleotides for Sequencing by Synthesis
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Solution Overview
Problem
Current nucleotide sequencing methods using 3′-allyl blocked nucleotides face challenges due to low efficiency of 3′ blocking group cleavage chemistry and rapid loss of signal intensity, limiting the application of sequencing by synthesis (SBS) for long DNA reads.
Innovation Solution
Development of nucleotides with 3′-OH acetal or thiocarbamate blocking groups that provide improved stability and efficiency in sequencing, using a Pd(0) catalyst for deblocking, and incorporation of detectable labels via cleavable linkers for enhanced sequencing metrics.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If 3'-allyl blocked nucleotides are used for sequencing by synthesis, then sequencing can be performed, but the 3' blocking group cleavage chemistry has low efficiency and signal intensity is rapidly lost
Solution Approach 1:
The patent changes the chemical structure of the 3' blocking group from allyl to acetal or thiocarbamate groups, which fundamentally alters the cleavage chemistry parameters. These new groups are designed to be cleaved by Pd(0) catalyst under mild conditions, achieving near-quantitative deblocking efficiency while maintaining sequencing reliability
Solution Approach 2:
The patent introduces Pd(0) catalyst as an intermediary substance to mediate the cleavage reaction of the 3' blocking group. This catalyst enables efficient removal of the acetal or thiocarbamate groups without interfering with the DNA polymerase reaction or causing signal loss, resolving the contradiction between reliable sequencing and efficient deblocking
2Duration of action of stationary object
If 3'-allyl blocked nucleotides are used for sequencing by synthesis, then sequencing can be performed, but signal intensity is rapidly lost limiting long DNA reads
Solution Approach 1:
The patent changes the fluorescent label chemistry by using novel fluorophores with enhanced photostability and quantum yield. These modified labels maintain signal intensity over extended sequencing cycles, enabling long read lengths without rapid signal decay
Solution Approach 2:
The patent uses Pd(0) catalyst as an intermediary that enables clean removal of the 3' blocking group without causing collateral damage to the fluorescent label or DNA structure. This selective deblocking prevents signal loss and maintains signal intensity throughout long sequencing runs
3Manufacturing precision
If conventional 3' blocking groups are used, then nucleotide incorporation can be controlled, but pre-phasing occurs due to incomplete deblocking
Solution Approach 1:
The patent changes the chemical properties of the 3' blocking group to acetal or thiocarbamate structures that are specifically designed for complete and uniform cleavage by Pd(0) catalyst. This ensures that all blocked nucleotides are efficiently deblocked at the same rate, eliminating pre-phasing while maintaining precise incorporation control
Solution Approach 2:
The Pd(0) catalyst acts as an intermediary that mediates uniform deblocking of all 3' blocked nucleotides in the population. This catalyst ensures synchronous removal of blocking groups, preventing the differential deblocking that causes pre-phasing and maintains sequencing accuracy
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 new nucleotides offer superior stability and deblocking rates, reducing pre-phasing and signal decay, enabling longer sequencing reads and improved data quality.
Implementation Method 1
using a Pd(0) catalyst for deblocking
Data Source
AI summary
Embodiments of the present disclosure relate to nucleotide and nucleoside molecules with 3′ acetal, thiocarbamate or allyl blocking groups. Also provided herein are methods to prepare such nucleotide and nucleoside molecules, and the uses of fully functionalized nucleotides containing the 3′-OH blocking group for sequencing applications.