Multimeric Fluorescent Reagents with Shield Elements
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Solution Overview
Problem
Fluorescent probes 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 DNA sequencing.
Innovation Solution
Development of multimeric protected fluorescent reagents with a shield element that limits contact between the fluorescent dye and binding elements, reducing photodamage and incorporating a shield element between the fluorescent dye and binding moiety to minimize interactions with enzymes, thereby protecting them from photodegradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent probes are used for real-time detection in DNA sequencing reactions, then sensitivity and selectivity of the signal are improved, but photodegradation of probes and photodamage to enzymes occur during extended exposure to excitation radiation
Solution Approach 1:
A shield element is introduced as an intermediary component between the fluorescent dye and the nucleotide binding element. This shield element physically separates the dye from the enzyme, preventing direct contact and photodamage while allowing the fluorescent signal to be detected for sequencing applications.
Solution Approach 2:
The fluorescent reagent is segmented into distinct functional components: a fluorescent dye portion for detection, a binding element for nucleotide recognition, and a shield element that separates them. This segmentation allows each component to perform its function independently without interfering with others.
2Productivity
If fluorescent dyes are positioned close to enzymes for detection during incorporation events, then real-time detection capability is improved, but enzyme inactivation occurs due to interactions with excited dyes
Solution Approach 1:
The shield element serves as a mediator that maintains the necessary proximity for detection while preventing harmful direct interactions. It allows the fluorescent signal to be generated and detected in real-time during incorporation events without the enzyme coming into direct contact with the excited dye.
Solution Approach 2:
The shield element is pre-positioned between the dye and enzyme to prevent harmful interactions before they can occur. This preliminary protective measure ensures enzyme stability throughout the sequencing reaction while maintaining detection capability.
3Duration of action of moving object
If traditional fluorescent reagents are used in highly processive reactions with extended excitation, then sequencing coverage is improved, but photodamage to reagents and components increases
Solution Approach 1:
The shield element continuously protects both the enzyme and fluorescent dye from harmful photodamage during extended sequencing reactions. This intermediary structure enables longer sequencing reads by preventing the accumulation of photodamage that would otherwise limit reaction duration.
Solution Approach 2:
The shield element provides beforehand protection against photodamage by physically separating the vulnerable components (enzyme and dye) before harmful interactions can occur. This protective barrier is in place from the beginning of the reaction, enabling sustained sequencing activity over longer periods.
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 exhibit improved stability and reduced photodamage, allowing for longer sequencing read lengths and increased accuracy in DNA sequencing reactions by shielding the fluorescent dyes from enzymes, thus maintaining enzyme activity and reaction integrity.
Implementation Method 1
fluorescent probes may be damaged during the course of the reaction or may inflict damage on other components of the reaction mixture
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
Multimeric protected fluorescent reagents and their methods of synthesis are provided. The reagents 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 reagents 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. The reagents also contain coupling elements connect monomeric compounds into multimeric forms, thereby increasing brightness.