Protected Fluorescent Reagent Shielding Enzymes
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Fluorescent reagents used in analytical systems are prone to photodegradation and can cause damage to enzymes and other components during extended exposure to excitation radiation, leading to compromised sequencing reactions in processes like single-molecule real-time sequencing.
Innovation Solution
Development of protected fluorescent reagent compounds with a multivalent central core element and shield elements that reduce contact between the fluorescent dye and binding elements, minimizing photodamage by incorporating a shield element between the fluorescent dye and the binding element, and using side chains with molecular weights of at least 300 to create a protective shell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent probes are used for real-time detection in sequencing reactions, then detection sensitivity and selectivity are improved, but photodamage to enzymes and other components increases due to extended exposure to excitation radiation
Solution Approach 1:
The patent introduces a protective group as an intermediary element between the fluorescent probe and the enzyme. This protective group absorbs or shields harmful excitation radiation, preventing direct interaction between the excited fluorescent probe and the enzyme, thereby reducing photodamage while maintaining detection sensitivity
Solution Approach 2:
The patent applies beforehand cushioning by incorporating protective groups into the fluorescent probe structure before the sequencing reaction begins. These protective groups pre-establish a shield against photodamage, allowing the system to withstand extended excitation radiation exposure during real-time sequencing without compromising enzyme activity
2Duration of action of moving object
If traditional fluorescent reagents are used for extended periods, then sequencing reaction coverage is improved, but photodegradation of the fluorescent probes increases
Solution Approach 1:
The protective groups are incorporated into the fluorescent probe structure before use, providing pre-established protection against photodegradation. This allows the fluorescent probes to maintain their chemical composition and fluorescence properties throughout extended sequencing reactions
Solution Approach 2:
The patent creates composite fluorescent probe structures by combining traditional fluorescent moieties with protective groups. This composite structure leverages the fluorescence properties of the original probe while adding the protective characteristics of the shield elements, achieving both long-duration functionality and enhanced stability
3Power
If fluorescent dyes are positioned close to enzymes for detection, then signal intensity is improved, but contact between fluorescent dye and enzyme increases causing inactivation
Solution Approach 1:
The protective group serves as a physical intermediary barrier between the fluorescent dye and the enzyme. It allows the fluorescent probe to remain positioned near the enzyme for strong signal detection while preventing direct contact that would cause enzyme inactivation, thus maintaining both signal intensity and enzyme reliability
Solution Approach 2:
The protective group acts as a flexible protective shell or thin film surrounding the fluorescent dye. This shell maintains close proximity to the enzyme for detection purposes while providing a physical barrier that prevents harmful interactions, balancing signal intensity with enzyme protection
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 protected fluorescent reagents significantly reduce photodamage to enzymes and other components, allowing for longer sequencing read lengths and improved accuracy by preventing contact between the fluorescent dye and enzymes, thus enhancing the stability and performance of fluorescent analytical systems.
Implementation Method 1
the fluorescent probes may be damaged during the course of the reaction or may inflict damage on other components of the reaction mixture. Such damage is particularly problematic in highly processive reactions, where the reaction mixture may be exposed to excitation radiation for extended periods of time
Implementation Method 2
the use of fluorescent optical signals in analytical systems is extremely powerful due to the sensitivity and selectivity of the signal
Data Source
AI summary
Protected fluorescent reagent compounds and their methods of synthesis are provided. The compounds are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The compounds contain fluorescent dye elements, that allow the compounds to be detected with high sensitivity at desirable wavelengths, binding elements, that allow the compounds to be recognized specifically by target biomolecules, and protective shield elements, that decrease undesirable contacts between the fluorescent dye elements and the bound target biomolecules and that therefore decrease photodamage of the bound target biomolecules by the fluorescent dye elements.