Multi-Component Probe System for Fluorescence Signal Amplification
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Solution Overview
Problem
Current DNA and RNA sequencing methods face challenges in generating strong fluorescence signals for reliable detection of single nucleotides, often resulting in weak signals overwhelmed by background noise, particularly in droplet-based sequencers.
Innovation Solution
A method involving progressive enzymatic digestion to generate a stream of single nucleoside triphosphates, using a multi-component probe system with a primary probe containing a restriction endonuclease nicking site and secondary probes with fluorophores in an undetectable state, which are activated through exonucleolytic digestion to produce a strong fluorescence signal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If a single-component probe system is used for nucleotide detection, then the device complexity is low, but the fluorescence signal intensity is weak and overwhelmed by background noise
Solution Approach 1:
The probe system is divided into multiple functional components: a first oligonucleotide with fluorophore and quencher, a second oligonucleotide for hybridization, and a third oligonucleotide for exonuclease resistance. This segmentation allows each component to perform its specific function while collectively generating a strong fluorescence signal through sequential assembly and enzymatic processing.
Solution Approach 2:
The probe components are designed to nest together through hybridization, with the second oligonucleotide binding to the first oligonucleotide, and the third oligonucleotide providing protective structure. This nested arrangement concentrates multiple fluorophores in close proximity to the target nucleotide, amplifying the fluorescence signal while maintaining structural organization.
2Measurement precision
If progressive enzymatic digestion is used to generate single nucleoside triphosphates, then the sequencing accuracy is improved, but the processing time increases
Solution Approach 1:
The probe system is pre-assembled with all necessary components (fluorophore-labeled oligonucleotide, hybridization partner, and exonuclease-resistant element) before encountering the target nucleotide. This preliminary preparation allows rapid detection once the nucleotide is generated, reducing the time penalty associated with progressive enzymatic digestion while maintaining high sequencing accuracy.
Solution Approach 2:
The mechanical/enzymatic process of progressive digestion is complemented by a chemical detection mechanism using fluorophore-quencher pairs and exonuclease-based signal amplification. This substitution of detection methodology allows parallel processing of multiple nucleotides without proportionally increasing total processing time, thereby improving efficiency while preserving accuracy.
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 significantly enhances the fluorescence signal intensity, improving the sensitivity and reliability of nucleotide detection, allowing for accurate sequencing and analysis of nucleic acids.
Implementation Method 1
each of which can be chemically and/or enzymatically manipulated to reveal the particular single nucleotide it originally contained. In one embodiment, these chemical and/or enzymatic manipulations comprise a method involving the use of one or more two-component oligonucleotide probe types each of which is adapted to be able to selectively capture one of the single nucleotide types from which the analyte is constituted. Typically, in each of such probe types, one of the two oligonucleotide components comprises characteristic fluorophores and in the probe's unused state the ability of these fluorophores to fluoresce remains extinguished by virtue of the presence of quenchers located close-by or by self-quenching. In use, when the probe has captured its corresponding single nucleotide, it is rendered susceptible to subsequent exonucleolysis thereby liberating the fluorophores from the quenchers and/or each other enabling them to fluoresce freely.
Data Source
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AI summary
A method of sequencing a nucleic acid is provided. It is characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (2) producing at least one substantially double-stranded primary oligonucleotide used probe by reacting, in the presence of a polymerase and a ligase, at least one of the single nucleoside triphosphates with a corresponding primary probe comprising (a) a first single- stranded oligonucleotide including a restriction endonuclease nicking-site, a single nucleotide capture site for capturing the single nucleoside triphosphate and oligonucleotide flanking regions juxtaposed either side of the capture site and (b) second and third single-stranded oligonucleotides capable of hybridising to the first oligonucleotide flanking regions; (3) nicking the first oligonucleotide strand of the used primary probe at the nicking-site with a nicking restriction endonuclease to create separate first oligonucleotide components; (4) separating the first oligonucleotide components generated in step (3) from the complementary strand of the used probe; (5) producing at least one substantially double-stranded secondary used probe by reacting, in the presence of a ligase, at least one of the separated first oligonucleotide components with a corresponding secondary probe comprising (c) a complementary fourth oligonucleotide bearing fluorophores in a substantially undetectable state and optionally (d) a fifth oligonucleotide at least in part complementary to the fourth oligonucleotide; (6) digesting the used secondary probe with an enzyme having double-stranded exonucleolytic activity to yield the fluorophores in a detectable state and a single-stranded sixth oligonucleotide which is at least in part the sequence complement of the fourth oligonucleotide and (7) detecting the fluorophores released in step (6). The method is advantageously carried out in microdroplets. Corresponding biological probe systems comprised of the primary and secondary probes are also described.