Reversible 3'-O Blocking Nucleoside Analogues for Sequencing

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Solution Overview

Problem

Current nucleic acid sequencing methods lack efficient, low-cost, high-throughput solutions for accurately sequencing and re-sequencing nucleic acids, particularly in terms of incorporating reversible 3′-O blocking groups and affinity tags for enhanced detection and analysis.

Innovation Solution

Development of nucleoside analogues with reversible 3′-O blocking groups and affinity tags linked via cleavable linkers, allowing for specific labeling and detection during sequencing by synthesis or ligation methods, utilizing polymers and enzymes to incorporate and detect these analogues within nucleic acid sequences.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If nucleoside analogues with reversible 3'-O blocking groups and affinity tags are used, then sequencing accuracy and detection capability are improved, but manufacturing complexity and cost increase

Engineering Contradiction:

Improvesequencing accuracyVSAvoidmanufacturing complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The nucleoside analogue is divided into distinct functional segments: a reversible 3'-O blocking group (R1) that controls polymerization, a nucleobase (R2) for template matching, a cleavable linker (L) that connects the nucleobase to the affinity tag, and an affinity tag (A1) for detection. This segmentation allows each component to perform its specific function independently, enabling precise control over the sequencing process while simplifying the overall manufacturing approach.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent employs reversible blocking groups that can be chemically modified between blocked and unblocked states. The blocking group R1 can be attached to or removed from the 3'-O position of the nucleoside analogue through chemical reactions, allowing dynamic control of polymerization activity. This parameter change enables sequential incorporation of nucleosides without permanent blocking, improving sequencing accuracy while using well-established chemical modification techniques.

Inventive Principle:

Principle #35Parameter changes

2Difficulty of detecting and measuring

If nucleoside analogues with affinity tags and detectable labels are incorporated, then detection capability is improved, but the complexity of incorporation and detection processes increases

Engineering Contradiction:

Improvedetection capabilityVSAvoidincorporation process complexity

Core Design Contradiction:

Difficulty of detecting and measuringVSDevice complexity

Solution Approach 1:

The nucleoside analogue integrates multiple functions into a single molecule: it serves as a polymerization substrate through the nucleobase, provides controlled reactivity through the reversible blocking group, enables detection through the affinity tag, and facilitates release through the cleavable linker. This multi-functionality is achieved using standard biochemical components and reactions, avoiding the need for complex specialized systems while significantly improving detection capability.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The cleavable linker L acts as an intermediary between the nucleobase and the affinity tag. It allows the affinity tag to be incorporated along with the nucleoside during polymerization, then subsequently removed under specific conditions to release the detectable label. This intermediary approach simplifies the detection process by using a dedicated removal step rather than requiring complex integrated systems.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Ease of operation

If reversible blocking groups are used to control nucleoside incorporation, then sequencing control is improved, but the duration of blocking and cleavage processes increases

Engineering Contradiction:

Improvesequencing controlVSAvoidblocking process duration

Core Design Contradiction:

Ease of operationVSDuration of action of moving object

Solution Approach 1:

The sequencing process uses periodic action through reversible blocking groups that are alternately attached and removed in cycles. Each nucleoside incorporation step is followed by a blocking step, then a detection step, and finally a cleavage step to remove the blocking group. This periodic cycle provides precise control over each sequencing step while using efficient chemical reactions that minimize the duration of each phase, maintaining overall process speed.

Inventive Principle:

Principle #19Periodic 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

Enables efficient and accurate nucleic acid sequencing by facilitating the reversible incorporation and detection of nucleoside analogues, improving sequencing throughput and reducing costs through targeted labeling and cleavage processes.

Implementation Method 1

R1 is a reversible blocking group selected from the group consisting of allenyl, cyanoethyl, cyanoethenyl, formaldehyde oximyl, acrylaldehyde oximyl, propionaldehyde oximyl, and cyanoethenaldehyde oximyl

Methodology Applied

Scientific EffectChemical Bonding: Chemical Bonding