3'-OH Blocking Groups for Stable Sequencing Nucleotide Incorporation
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Solution Overview
Problem
Existing nucleotide sequencing technologies face challenges in achieving stable and efficient incorporation of 3'-hydroxy protecting groups that prevent additional nucleotide additions while maintaining the integrity of the polynucleotide chain, and require conditions for removal that do not damage the DNA.
Innovation Solution
Development of nucleotides with 3'-OH acetal or thiocarbamate blocking groups that offer improved stability during synthesis and sequencing, allowing for efficient incorporation and removal under mild conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a 3'-hydroxy protecting group is added to prevent additional nucleotide incorporation, then sequencing accuracy is improved, but the stability of the protecting group during synthesis and sequencing becomes problematic
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of the protecting group from conventional options to acetal or thiocarbamate groups. This structural parameter change enables the group to maintain stability during synthesis and sequencing while still allowing controlled removal under mild conditions, thus resolving the contradiction between needing the group to be stable yet removable.
2Productivity
If a protecting group is designed to be easily removable, then sequencing cycle efficiency is improved, but the risk of damaging the polynucleotide chain increases
Solution Approach 1:
The patent changes the chemical parameters of the protecting group to acetal or thiocarbamate structures, which can be removed under mild conditions (such as mild acid or enzymatic conditions) that do not damage the polynucleotide chain. This enables efficient deprotection while maintaining chain integrity, resolving the contradiction between removal ease and chain safety.
3Ease of manufacture
If conventional protecting groups are used, then synthesis is simpler, but pre-phasing occurs leading to reduced data quality
Solution Approach 1:
The patent modifies the protecting group structure to acetal or thiocarbamate, which exhibit lower pre-phasing behavior during sequencing. This structural parameter change improves data quality by reducing sequencing errors while the groups remain compatible with standard synthesis protocols, thus resolving the contradiction between synthesis simplicity and data quality.
Data Source
Figure 1
Figure 2A~2B
Figure 3A~3B
AI summary
Embodiments of the present disclosure relate to nucleotide and nucleoside molecules with acetal or thiocarbamate 3ʹ-OH blocking groups. Also provided herein are methods to prepare such nucleotide and nucleoside molecules, and the uses of fully functionalized nucleotides containing the 3ʹ-OH blocking group for sequencing applications.