Cleavable Fluorescent Reversible Terminators for Accurate DNA Sequencing

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Solution Overview

Problem

Current DNA sequencing methods, particularly sequencing by synthesis (SBS), face challenges in accurately detecting nucleotide incorporation in homopolymeric regions and are hindered by the sensitivity of DNA polymerase to modifications at the 3′ position, leading to inaccuracies and prolonged detection times.

Innovation Solution

The method involves using nucleotide analogues with a unique fluorescent label linked via a cleavable linker to the 3′-oxygen of the deoxyribose and a small moiety to cap the 3′-OH group, allowing incorporation by DNA polymerase, followed by removal of the label and moiety to regenerate the 3′-OH for subsequent reactions, enabling accurate and efficient sequencing.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If a fluorescent label is attached to the 3'-position of the nucleotide for detection, then sequencing detection capability is improved, but DNA polymerase activity is inhibited due to sensitivity to 3'-position modifications

Engineering Contradiction:

Improvenucleotide detection accuracyVSAvoidDNA polymerase activity

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The fluorescent label is separated from the 3'-position by inserting a linker arm between them. This segmentation allows the label to be positioned away from the critical 3'-OH group, maintaining polymerase activity while enabling fluorescence detection of the incorporated nucleotide.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A linker arm acts as an intermediary component connecting the fluorescent label to the nucleotide base. This intermediary structure transmits the detection signal while physically separating the label from the 3'-position, preventing interference with polymerase function.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Measurement precision

If each nucleotide is added and detected separately to ensure accurate identification, then measurement precision is improved, but the overall detection time increases

Engineering Contradiction:

Improvenucleotide identification accuracyVSAvoiddetection time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

All four fluorescently labeled nucleotide analogues (dATP, dCTP, dGTP, dTTP) are combined in a single reaction mixture and added simultaneously to the sequencing reaction. The fluorescent labels with different emission wavelengths allow parallel detection of all incorporated nucleotides in one step, rather than sequential addition and detection.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

Different fluorescent labels with distinct emission spectra (colors) are assigned to each nucleotide type. This color-coding system enables simultaneous identification of multiple nucleotides through their unique fluorescence signatures, achieving parallel detection without sacrificing accuracy.

Inventive Principle:

Principle #32Color changes

3Measurement precision

If the fluorescent label remains attached to the nucleotide for continuous detection, then measurement capability is maintained, but the accuracy decreases due to accumulation of un-degraded nucleotides

Engineering Contradiction:

Improvesequencing accuracyVSAvoidaccumulation of un-degraded nucleotides

Core Design Contradiction:

Measurement precisionVSQuantity of substance

Solution Approach 1:

The fluorescent label is periodically removed from the nucleotide after detection through controlled degradation. This periodic removal prevents accumulation of labeled nucleotides that could interfere with subsequent cycles, while maintaining the ability to detect incorporated nucleotides in each cycle.

Inventive Principle:

Principle #19Periodic action

Solution Approach 2:

The fluorescent label is discarded from unincorporated nucleotides through selective degradation, removing them from the reaction mixture to prevent interference. The labeling system allows for recovery and reuse of the nucleotide bases after label removal.

Inventive Principle:

Principle #34Discarding and recovering

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 enables accurate sequencing of DNA, particularly in homopolymeric regions, with increased throughput and reduced detection time, as it allows for parallel sequencing of multiple DNA molecules on a chip using 4-color fluorescence imaging.

Implementation Method 1

each nucleotide analogue comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, and analogues thereof, (ii) a deoxyribose, (iii) a moiety cleavably linked to the 3'-oxygen of the deoxyribose and (iv) a unique label cleavably linked to the base

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS12559797B2Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

Publication Date: 2026.02.24 THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK

AI summary

This invention provides a process for sequencing single-stranded DNA by employing a nanopore and modified nucleotides.