Polymerase-Luciferase Sequencing for Autofluorescence-Free Base Calling
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Solution Overview
Problem
Conventional nucleic acid sequencing methods face challenges such as autofluorescence and incomplete cleavage of fluorescent labels, leading to reduced sensitivity and signal intensity, particularly in cell or tissue samples.
Innovation Solution
The use of polymerase-luciferase fusion proteins that emit distinguishable luminescent signals without the need for excitation light, combined with non-fluorescent nucleotides and non-reversible terminators, to stabilize ternary complexes and detect nucleotide incorporation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent labels are used to detect nucleotide incorporation, then signal detection is enabled, but autofluorescence and incomplete cleavage reduce sensitivity and accuracy
Solution Approach 1:
The patent replaces the optical detection system (fluorescence) with a luminescence-based detection system. Instead of using fluorophores that require excitation light and suffer from autofluorescence interference, the invention uses polymerases fused to luciferase enzymes that emit luminescent signals directly. This substitution eliminates the need for excitation light sources and removes the harmful autofluorescence effect entirely, while maintaining the ability to detect nucleotide incorporation events with high precision.
Solution Approach 2:
The patent changes the detection parameter from fluorescent signal intensity to luminescence signal intensity. By using polymerase-luciferase fusion proteins, the detection system measures luminescence emitted by the fusion protein when it forms a ternary complex with the template and nucleotide. This parameter change from fluorescence to luminescence fundamentally removes the autofluorescence interference problem while preserving the ability to distinguish different nucleotide incorporations through distinguishable luminescent signals.
2Measurement precision
If reversible terminator nucleotides are used for sequencing, then base identification is enabled, but incomplete cleavage leads to phasing errors
Solution Approach 1:
The patent extracts and removes the reversible terminator group from the nucleotide structure. Instead of using nucleotides with reversible terminators that require cleavage steps, the invention employs nucleotides without terminators. This extraction of the terminator component eliminates the source of incomplete cleavage problems and phasing errors, while the sequencing process continues to rely on the natural incorporation properties of unmodified nucleotides.
Solution Approach 2:
The patent uses disposable, non-reversible nucleotide incorporation events. Each nucleotide incorporation is detected through the luminescent signal from the polymerase-luciferase fusion protein, and the nucleotide remains permanently incorporated without requiring reversible termination or cleavage. This approach treats each nucleotide as a single-use element that provides its signaling function upon incorporation and then remains in the sequence, eliminating the need for complex reversible/irreversible switching mechanisms.
3Difficulty of detecting and measuring
If excitation light is used to detect fluorescent signals, then nucleotide incorporation can be detected, but autofluorescence from cell or tissue samples interferes with detection
Solution Approach 1:
The patent substitutes the fluorescence detection mechanism with a luminescence detection mechanism. Instead of using fluorophores that require excitation light to emit signals, the invention employs polymerase-luciferase fusion proteins that directly emit luminescent signals upon substrate interaction. This mechanical substitution eliminates the need for excitation light sources and removes the autofluorescence interference that plagues fluorescence-based methods, particularly in complex biological samples like cells and tissues.
Solution Approach 2:
The patent converts the limitation of requiring excitation light into a benefit by using luminescence instead. The harmful autofluorescence background that arises from excitation light interaction with biological samples is completely avoided because luminescence does not require excitation light. The polymerase-luciferase fusion proteins generate their own detectable signals through enzymatic reaction with substrate, turning the detection process into a self-luminescent event that is inherently free from autofluorescence interference.
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 enhances sequencing sensitivity and accuracy by avoiding autofluorescence and incomplete cleavage issues, allowing for precise base identification in nucleic acid sequencing, especially in complex biological samples.
Implementation Method 1
polymerases labeled with luciferase proteins capable of emitting distinguishable luminescent signals in the presence of substrate
Data Source
AI summary
The present disclosure relates in some aspects to methods, systems, and kits for sequencing a template nucleic acid molecule using one or more polymerase-luciferase fusion proteins. In some embodiments, different types of polymerase-luciferase fusion proteins and are sequentially contacted with a template nucleic acid in the presence of different types of nucleotides under conditions that stabilize a ternary complex between a given polymerase-luciferase fusion protein, the template nucleic acid molecule, and a nucleotide when the nucleotide is complementary to the template nucleic acid. By imaging the sample to detect luminescence, the base of the template nucleic acid can be identified.