Thiolated Nucleotide Analogues for Hydrolysis-Resistant Sequencing
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Solution Overview
Problem
Current nucleic acid synthesis methods face inefficiencies due to nucleotide polyphosphates being susceptible to hydrolysis by phosphatase and pyrophosphatase enzymes, which can hinder the nucleotide incorporation process and reduce the effectiveness of sequencing reactions, especially in miniaturized formats where signal detection is compromised.
Innovation Solution
The use of sulfur-containing thiolated nucleotides, such as deoxyribonucleotide-5′-γ-thio-triphosphates, which are resistant to hydrolysis by phosphatase and pyrophosphatase enzymes, allowing for efficient incorporation into nucleic acids and maintaining reaction efficiency even in miniaturized formats by preventing unwanted hydrolysis and maintaining signal detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If standard nucleotide polyphosphates are used for nucleic acid synthesis, then the nucleotide incorporation process can proceed, but the nucleotides are susceptible to hydrolysis by phosphatase and pyrophosphatase enzymes, reducing reaction effectiveness
Solution Approach 1:
The patent applies parameter changes by substituting one oxygen atom in the phosphate group with a sulfur atom, creating a thiophosphate moiety. This chemical parameter change fundamentally alters the nucleotide's resistance to hydrolysis by phosphatase and pyrophosphatase enzymes, while maintaining its ability to be incorporated into nucleic acids by polymerase enzymes. The sulfur substitution preserves the nucleotide's functional properties while conferring enzymatic resistance.
2Productivity
If miniaturized formats are used for sequencing reactions, then reaction efficiency can be improved, but signal detection is compromised due to hydrolysis of nucleotide polyphosphates
Solution Approach 1:
The thiophosphate modification changes the chemical stability parameter of the nucleotide, preventing hydrolysis that would otherwise occur in miniaturized reaction formats. This allows the reaction system to maintain both high efficiency and accurate signal detection in miniaturized sequencing applications.
3Productivity
If thiolated nucleotides are used to prevent hydrolysis, then reaction efficiency is maintained, but the nucleotide structure is modified
Solution Approach 1:
The patent implements a minimal structural modification by replacing only one oxygen atom with sulfur in the phosphate group, rather than extensively modifying the nucleotide structure. This subtle parameter change achieves the desired enzymatic resistance while preserving the nucleotide's natural incorporation properties and maintaining high reaction efficiency.
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
Thiolated nucleotides ensure high incorporation rates comparable to non-thiolated counterparts and resist hydrolysis, enhancing the efficiency and yield of nucleotide incorporation reactions, thereby improving sequencing reaction efficiency and read-length in miniaturized formats.
Implementation Method 1
the at least one nucleotide is resistant to hydrolysis by phosphatase
Implementation Method 2
incorporating a nucleotide residue at a terminus of a nucleic acid using a polymerase enzyme
Data Source
AI summary
The present disclosure provide systems, compositions, methods, reagents, kits and products for extending a nucleic acid that includes incorporating a nucleotide residue at a terminus of a nucleic acid using a polymerase enzyme and at least one nucleotide, wherein the at least one nucleotide includes a thiophosphate moiety, and wherein the at least one nucleotide is resistant to hydrolysis by phosphatase. In some embodiments, the nucleotide incorporation can be conducted in the presence of a phosphatase. In some embodiments, the nucleotide incorporation can be conducted in the presence of at least on chelation moiety that is configured to bind an orthophosphate moiety.