Cleavable Nucleotide Analogues for Scarless DNA Sequencing
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Solution Overview
Problem
Current DNA sequencing methods, particularly sequencing by synthesis (SBS), face challenges due to the accumulation of 'scars' at the nucleotide bases, which interfere with DNA polymerase recognition and limit read length.
Innovation Solution
Development of nucleotide analogues with a 3′-O blocking group that is cleavable under mild conditions, allowing for the incorporation of scarless nucleotides into growing DNA chains, and the use of thermophilic nucleic acid polymerase complexes with these analogues to enhance sequencing efficiency and read length.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cleavable fluorescent NRTs with 3′-O blocking groups are used for SBS sequencing, then fluorescent signal detection is enabled, but residual scars accumulate at nucleotide bases interfering with DNA polymerase recognition
Solution Approach 1:
The patent extracts and removes the harmful scar moiety from the nucleotide structure after fluorescent dye cleavage. By designing the 3′-O blocking group to be cleavable along with the fluorescent dye, the residual scar is completely removed from the DNA chain, eliminating its interfering effect on subsequent polymerase reactions while maintaining fluorescent signal detection capability
Solution Approach 2:
The patent applies discarding and recovering by cleaving off both the fluorescent dye and the 3′-O blocking group together as a unified removable unit. This ensures that after sequencing detection, the nucleotide returns to its natural state without residual modifications, allowing continuous high-fidelity sequencing cycles
2Measurement precision
If 3′-O bulky-dye-modified nucleotides are used as substrates, then fluorescent labeling is achieved, but DNA polymerase has difficulty accepting them as substrates
Solution Approach 1:
The patent segments the nucleotide structure by placing the bulky fluorescent dye and 3′-O blocking group on separate functional domains. The 3′-O blocking group is designed to be cleavable, allowing the polymerase to initially accept the nucleotide substrate, then subsequently remove the bulky modification after incorporation, resolving the conflict between fluorescent labeling and polymerase acceptance
Solution Approach 2:
The patent applies preliminary action by first allowing the polymerase to incorporate the fluorescently labeled nucleotide substrate, then subsequently cleaving off the 3′-O blocking group and fluorescent dye. This sequence enables the polymerase to work with the modified substrate initially, then restores the natural nucleotide state after incorporation
3Length of moving object
If successive rounds of SBS are performed, then sequencing read length increases, but accumulated scars significantly interfere with DNA double helix structure
Solution Approach 1:
The patent enables continuous sequencing cycles by completely removing scars after each round. The 3′-O blocking group and fluorescent dye are cleaved together, restoring the natural 3′-OH terminus and eliminating structural interference, allowing the DNA double helix to maintain stability throughout multiple sequencing rounds
Solution Approach 2:
The patent achieves continuous sequencing by ensuring complete scar removal after each cycle. The cleavable 3′-O blocking group design allows uninterrupted sequencing reactions without accumulation of harmful residues, maintaining DNA structural integrity and polymerase activity across successive SBS rounds
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 proposed solution enables efficient and accurate incorporation of nucleotides into DNA sequences, reducing the interference of 'scars' and allowing for longer read lengths in DNA sequencing, thereby improving the accuracy and efficiency of sequencing processes.
Implementation Method 1
determines DNA sequences during the polymerase reaction using cleavable fluorescently labeled nucleotide reversible terminator (NRT) sequencing chemistry
Implementation Method 2
production of a small molecular 'scar' (e.g., a propargylamine or a modified propargylamino moiety) at the nucleotide base after cleavage of the fluorescent dye from the incorporated nucleotide
Data Source
AI summary
Disclosed herein, inter alia, are compounds, compositions, and methods of use thereof in the sequencing a nucleic acid.