Fluorescent Polymerase Substrates With Protein Shields
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Solution Overview
Problem
Current nucleic acid sequencing methods, particularly those involving polymerase-mediated template-dependent nucleic acid synthesis, face challenges with photostability, leading to reduced sequencing read lengths and efficiency due to enzyme damage from excitation radiation.
Innovation Solution
Development of a polymerase enzyme substrate with a shielding protein that separates the nucleotide and fluorescent dye components by a distance, preventing contact between the fluorescent dye and the polymerase enzyme, thereby mitigating photodamage and enhancing enzyme stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent dye is coupled directly to nucleotide for real-time detection, then detection sensitivity is improved, but enzyme photostability deteriorates due to direct contact between dye and polymerase enzyme
Solution Approach 1:
The substrate is segmented into distinct functional domains: a nucleotide component for enzyme binding and incorporation, a linker component for flexible connection, and a fluorescent dye component for detection. This segmentation allows the nucleotide to interact with the polymerase while the dye remains spatially separated, reducing photodamage to the enzyme.
Solution Approach 2:
A linker component acts as an intermediary between the nucleotide and fluorescent dye. This linker provides a flexible tether that allows the dye to be positioned away from the polymerase active site while still enabling detection of nucleotide incorporation events through fluorescence changes.
2Productivity
If fluorescent dye is positioned close to nucleotide active site, then real-time detection efficiency is improved, but enzyme damage from excitation radiation increases
Solution Approach 1:
The substrate exhibits local quality differentiation where the nucleotide portion is designed for high-affinity binding to the polymerase active site, while the fluorescent dye portion is positioned in a different spatial zone that allows detection without direct interaction with the enzyme. This local differentiation enables both high sequencing efficiency and reduced enzyme damage.
Solution Approach 2:
The fluorescent dye is positioned in a different spatial dimension relative to the nucleotide active site, using the linker to extend into the solution phase rather than remaining in the enzyme active site cleft. This dimensional separation allows the dye to report on incorporation events without being constrained to the same spatial location as the enzyme catalytic center.
3Stability of the object's composition
If nucleotide and dye are rigidly connected, then structural stability is improved, but flexibility in binding to polymerase active site deteriorates
Solution Approach 1:
The linker component introduces dynamic flexibility to the substrate structure. It allows the nucleotide and fluorescent dye to move relative to each other, enabling the nucleotide to adopt optimal binding conformations in the polymerase active site while the dye can move to minimize interference with enzyme function during catalysis.
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 results in significantly longer sequencing read lengths and improved photostability, allowing for more reliable and efficient nucleic acid sequencing by preventing direct contact between the fluorescent dye and the polymerase enzyme.
Implementation Method 1
a fluorescent dye moiety attached to a second position on the protein, wherein the first and second attachment points are spaced apart by a distance such that when a nucleoside phosphate attached to the protein is in the active site of the polymerase enzyme, a fluorescent dye moiety attached to the protein is shielded by the protein from coming into contact with the polymerase enzyme
Data Source
AI summary
Compositions, methods, and systems are provided for fluorescent polymerase enzyme substrates comprising protein shields for improving enzyme photostability in single molecule real time sequencing. Fluorescent polymerase enzyme substrates of the invention have a protein shield between the fluorescent dye moieties and nucleotide moieties of the polymerase enzyme substrate. The polymerase enzyme substrates have a nucleotide component and a dye component, each attached to a protein. The attachments can be covalent. The protein can, for example, prevent the direct interaction of the fluorescent dye moiety with the enzyme when carrying out nucleotide synthesis, preventing photodamage to the enzyme. The polymerase enzyme substrates of the invention can have multiple dyes and multiple nucleotide moieties.