Cleavable Nucleotide Linkers for Polymerase-Compatible DNA Sequencing
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Solution Overview
Problem
Existing DNA sequencing methods, particularly sequencing by synthesis (SBS), lack modified nucleotides and nucleosides that are effectively recognized by DNA polymerases and efficiently incorporated into growing DNA chains, hindering the goal of achieving the $1,000 genome.
Innovation Solution
Development of compounds with specific moieties that are recognized by DNA polymerases, incorporating detectable labels for sequencing, and using cleavable linkers to facilitate nucleic acid sequencing by synthesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If modified nucleotides and nucleosides are used to enable detectable labeling for sequencing, then sequencing capability is improved, but recognition efficiency by DNA polymerases deteriorates
Solution Approach 1:
The modified nucleotide is divided into distinct functional modules: a nucleobase portion that maintains polymerase recognition, a nucleoside portion for structural integrity, and a separately attached detectable label connected via a cleavable linker. This segmentation allows each component to perform its specialized function without interfering with polymerase recognition of the core nucleotide structure.
Solution Approach 2:
A cleavable linker acts as an intermediary between the nucleotide core and the detectable label. This intermediary allows the label to be attached for sequencing detection while providing a mechanism for its subsequent removal, preventing interference with DNA polymerase recognition and incorporation during the sequencing process.
2Measurement precision
If detectable labels are attached to nucleotides for sequencing detection, then measurement capability is improved, but incorporation efficiency into growing DNA chains deteriorates
Solution Approach 1:
The detectable label is preliminarily attached to the nucleotide via a cleavable linker before the sequencing reaction. This allows the labeled nucleotide to be incorporated into the DNA chain during normal sequencing, after which the label is removed in a subsequent step. The preliminary attachment enables detection without compromising incorporation efficiency during the critical sequencing step.
Solution Approach 2:
The chemical state of the nucleotide is dynamically changed: initially present with a detectable label attached via cleavable linker for detection purposes, then transformed after incorporation by removing the label through cleavage. This parameter change allows the system to optimize for detection during sequencing and for natural nucleotide behavior during incorporation.
3Measurement precision
If cleavable linkers are used to attach detectable labels, then sequencing accuracy is improved, but device complexity deteriorates
Solution Approach 1:
The detectable label is extracted as a separate, removable component from the nucleotide core through the use of a cleavable linker. This extraction allows the label to be attached for sequencing detection and then completely removed afterward, simplifying the final DNA structure and avoiding permanent complexity while maintaining sequencing accuracy during the measurement phase.
Data Source
AI summary
Disclosed herein, inter alia, are compounds, compositions, and methods of use thereof for sequencing a nucleic acid.