Detectable Nucleotide Analogues for Clean-Cleavage SBS Sequencing
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Solution Overview
Problem
Existing DNA sequencing methods face challenges in achieving long read lengths and efficient nucleotide detection, particularly in sequencing by synthesis (SBS) technologies, due to the stability and manipulation requirements of cleavable linkers in fluorescent nucleotide reversible terminators.
Innovation Solution
Development of nucleotide analogues with covalent linkers and detectable labels, such as 3′-O—SS(DTM)-dNTPs, that incorporate orthogonal binding molecules and cleavable moieties, allowing for precise nucleotide identification and regeneration of the 3′-OH group for continuous sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If cleavable fluorescent nucleotide reversible terminators are used for sequencing by synthesis, then nucleotide incorporation and signal detection can be achieved, but the cleavable linker requires stability during sequencing reactions and minimal manipulation while leaving no long tail on the base after cleavage
Solution Approach 1:
The patent modifies the chemical structure of the cleavable linker by changing parameters such as the cleavage mechanism (photocleavable vs. enzymatic), the linker length, and the chemical composition to achieve optimal stability during sequencing while enabling clean cleavage without residual tails on the base
Solution Approach 2:
The patent designs the cleavable linker to be a simplified copy or analog of natural DNA components, allowing it to function similarly to natural bases during polymerase recognition while providing controlled cleavage properties that natural bases lack
2Measurement precision
If fluorescent labels are attached to nucleotide bases for detection, then sequence determination is enabled, but the labels must not interfere with polymerase recognition and incorporation
Solution Approach 1:
The patent attaches fluorescent labels to specific positions on the nucleotide base structure (such as the C5 position of pyrimidines or C8 position of purines) rather than random attachment, ensuring that the label is positioned in a location that does not interfere with polymerase active site recognition while still providing detectable signal
Solution Approach 2:
The patent uses a linker or spacer arm as an intermediary between the fluorescent label and the nucleotide base, allowing the label to be positioned at an optimal distance from the base to minimize steric interference with polymerase binding while maintaining fluorescence detection capability
3Measurement precision
If 3′-OH capping moieties are used to terminate polymerase reaction for sequence determination, then single base identification per cycle is achieved, but the capping moiety must be efficiently removed to resume polymerase reaction in the next cycle
Solution Approach 1:
The patent incorporates the cleavable capping moiety as an integral part of the nucleotide analog structure before incorporation, so that the termination and subsequent cleavage functions are pre-built into the substrate itself, enabling efficient cycle progression without additional processing steps
Solution Approach 2:
The patent designs the capping moiety to be automatically removed through its inherent cleavable properties (photocleavable or enzymatically cleavable groups) after serving its termination function, allowing the 3′-OH group to be regenerated without complex external intervention and ready for the next incorporation cycle
Data Source
AI summary
Disclosed herein, inter alia, are compounds, compositions, and methods of use thereof in the sequencing a nucleic acid.