Nucleotide Cleavable Linkers with Rigid Spacers for Sequencing
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Solution Overview
Problem
Current DNA sequencing methods, particularly sequencing by synthesis (SBS), face challenges in accurately and efficiently incorporating modified nucleotides into growing DNA chains, which affects the accuracy and efficiency of sequencing reactions.
Innovation Solution
Development of compounds with specific structures, including a divalent nucleobase, polymerase-compatible cleavable linkers, and detectable moieties, that are incorporated into primers to create extension strands, allowing for accurate detection and sequencing of nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If modified nucleotides are incorporated into growing DNA chains during SBS, then sequencing accuracy and efficiency are improved, but the complexity of the sequencing reaction increases
Solution Approach 1:
The nucleotide is divided into distinct functional segments: a reversible terminator portion with detectable moiety for identification, a cleavable linker for controlled release, and a nucleobase portion for polymerase recognition. This segmentation allows each component to perform its specific function independently, improving sequencing accuracy while managing reaction complexity through modular design.
Solution Approach 2:
The cleavable linker acts as an intermediary between the reversible terminator and the nucleobase. It maintains the nucleotide in an inactive, blocked state during polymerization, then enables controlled activation through cleavage. This intermediary mechanism simplifies the overall reaction by providing a reliable switch between incorporation and release states.
2Measurement precision
If reversible terminators with detectable moieties are used, then compound identification accuracy is improved, but the number of reaction components increases
Solution Approach 1:
Multiple functions are merged into the reversible terminator structure: the detectable moiety provides identification, the cleavable linker provides controlled release, and the nucleobase portion provides polymerase recognition. This merging reduces the number of separate components needed in the reaction system while maintaining high identification accuracy through the integrated design.
Solution Approach 2:
The reversible terminator structure serves multiple purposes simultaneously: it acts as a polymerase substrate, a detectable label carrier, and a controlled-release mechanism. This multi-functionality reduces the overall number of reaction components needed, as a single molecule performs roles that would traditionally require separate elements.
3Productivity
If cleavable linkers are used to release incorporated nucleotides, then sequencing efficiency is improved, but the risk of premature cleavage increases
Solution Approach 1:
The cleavable linker is designed with specific chemical parameters that confer resistance to premature cleavage under normal polymerization conditions. The linker's structural and chemical properties are optimized to remain stable during DNA synthesis but become susceptible to controlled cleavage at the appropriate stage, ensuring reliable timing of nucleotide release without premature degradation.
Data Source
AI summary
Disclosed herein, inter alia, are compounds, compositions, and methods of use thereof for sequencing a nucleic acid.