3'-OH Blocked Nucleotides for Rapid Sequencing Reactivation

Solution Overview

Problem

Current nucleic acid sequencing technologies using dNTP analogues with reversible blocking groups face limitations in stability and efficiency, particularly with azido groups that can be unstable and pose safety risks, and lack efficient methods for rapid and controlled removal of blocking groups.

Innovation Solution

Development of dNTP analogues with azidomethyl as a 3'-OH reversible blocking group that undergoes a cascade reaction, including a Staudinger reaction and hydrolysis, allowing for rapid and controlled removal, and incorporation of ester or carbonate groups to enhance stability and reactivity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If azido group is used as reversible blocking group, then blocking efficiency is improved, but stability deteriorates and safety risks increase

Engineering Contradiction:

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The patent introduces phosphorous reagent as an intermediary that mediates the removal of azido group through Staudinger reaction. The azido group remains stable during sequencing but can be selectively removed by the phosphorous reagent, which acts as a controlled intermediary to convert the stable azido group into a reactive intermediate that rapidly hydrolyzes to free 3'-OH.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If direct blocking of 3'-OH is used, then blocking efficiency is improved, but removal speed deteriorates

Engineering Contradiction:

Core Design Contradiction:

ReliabilityVSSpeed

Solution Approach 1:

The patent implements continuous useful action through cascade reaction mechanism. The Staudinger reaction produces an intermediate with methylene amino group that immediately undergoes rapid hydrolysis to free 3'-OH. This continuous cascade ensures both stable blocking during sequencing and rapid removal when needed, maintaining efficient continuous operation.

Inventive Principle:

Principle #20Continuity of useful action

3Speed

If azido group is used for rapid removal, then removal speed is improved, but safety risks worsen

Engineering Contradiction:

Core Design Contradiction:

SpeedVSObject-affected harmful factors

Solution Approach 1:

The patent applies preliminary anti-action by using phosphorous reagent to preemptively convert the potentially hazardous azido group into a safe intermediate through Staudinger reaction. This preliminary conversion neutralizes the safety risks of azido group while maintaining its rapid removal capability, preventing harmful effects before they can occur.

Inventive Principle:

Principle #9Preliminary anti-action

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

The azidomethyl group provides stable and rapid removal of the blocking function, enabling efficient nucleic acid sequencing with improved accuracy and safety by ensuring precise termination and reactivation of nucleic acid synthesis.

Implementation Method 1

the azido group undergoes the Staudinger reaction under the action of phosphorous reagent to produce an intermediate with methylene amino group on 3'-OH

Methodology Applied

Scientific EffectStaudinger reaction:

Implementation Method 2

which then undergoes a rapid hydrolysis reaction to free the 3'-OH

Methodology Applied

Scientific Effecthydrolysis reaction: Hydrolysis

Data Source

PatentEP4234566B1Modified nucleoside or nucleotide

Publication Date: 2026.04.15

Figure 1~3

AI summary

A modified nucleoside or nucleotide, the 3'-OH of the modified nucleoside or nucleotide being reversibly blocked; meanwhile, the present invention also relates to a kit comprising the nucleoside or nucleotide, and a sequencing method based on the nucleoside or nucleotide.