3'-Nitrate Nucleotides for Sequencing by Synthesis
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Solution Overview
Problem
Current nucleic acid sequencing methods using reversibly blocked nucleotides face challenges with incomplete blockage due to residual esterase activities, leading to sub-optimal performance in quantitative enzymatic incorporation and efficient deprotection.
Innovation Solution
Development of deoxynucleoside tri- or tetraphosphates with a 3'-nitrate blocking group and a detectable fluorescent label linked to the nucleobase, using a cleavable linker, and the inclusion of pyrophosphatase in polymerase-catalyzed DNA synthesis reactions to stabilize the 3'-nitrate blocking entity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If esters and ethers are used as 3'-protecting groups in nucleoside triphosphates, then the nucleotides can be reversibly blocked for sequencing by synthesis, but residual esterase activities cause incomplete blockage leading to sub-optimal performance
Solution Approach 1:
The patent changes the chemical parameter of the protecting group from esters/ethers to nitrate at the 3'-position. This parameter change eliminates susceptibility to esterase hydrolysis while maintaining reversible blockage capability through alternative deprotection mechanisms (e.g., photolysis or chemical reduction), thereby achieving complete and reliable blockage.
2Measurement precision
If a fluorescent label is attached to the nucleobase via a cleavable linker for detection, then sequencing detection is enabled, but the additional structural complexity increases synthesis difficulty
Solution Approach 1:
The patent combines the fluorescent label attachment to the nucleobase directly through a cleavable linker in a modular fashion. The linker serves dual purposes: enabling fluorescent detection during sequencing and providing a controlled release mechanism for the nucleobase after incorporation. This merging of detection and release functions into a single structural element simplifies the overall synthesis process compared to adding separate components.
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
This approach ensures complete and stable blockage of the 3' terminus, improving the efficiency and accuracy of nucleotide incorporation and deprotection, thereby enhancing the performance of DNA sequencing reactions.
Implementation Method 1
the inclusion of pyrophosphatase in polymerase-catalyzed DNA synthesis reactions to stabilize the 3'-nitrate blocking entity
Implementation Method 2
a detectable fluorescent label linked to the nucleobase, using a cleavable linker
Data Source
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AI summary
The present invention provides deoxynucleoside tri - or tetraphosphate comprising a 3' nitrate and a detectable label covalently bound to the oxygen atom of a oxymethyl or oxyallyl or oxypropargyl substitution of a nucleobase. Such compounds provide new possibilities for future Sequencing by Synthesis technologies.