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DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

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Towards a fluorescent molecular switch for nucleic acid biosensing.

Melissa Massey¹, Ulrich J Krull

¹Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada.

Analytical and Bioanalytical Chemistry

|May 15, 2010

Summary

A new fluorescent molecular switch detects nucleic acid hybridization using self-assembled Neutravidin and thiazole orange dye. This solid-phase sensor shows a fivefold fluorescence increase upon target binding, enabling sensitive DNA detection.

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Area of Science:

Biochemistry
Molecular Biology
Nanotechnology

Background:

Development of sensitive nucleic acid detection methods is crucial for diagnostics.
Solid-phase analysis offers advantages for sensor immobilization and regeneration.
Fluorescent molecular switches provide a platform for signal transduction in biosensing.

Purpose of the Study:

To design and characterize a novel fluorescent molecular switch for nucleic acid hybridization detection.
To develop a sensor amenable for solid-phase analysis using immobilized components.
To investigate the factors influencing the switch's performance and signal output.

Main Methods:

Self-assembly of biotinylated components (Neutravidin, linker with thiazole orange dye, probe oligonucleotide) onto Neutravidin.
Immobilization of the switch constructs onto biotin-functionalized optical fibers.
Fluorescence intensity and fluorescence lifetime measurements to assess hybridization events.
Optimization of solution conditions (pH, ionic strength) to enhance dye binding modes.

Main Results:

Successful construction of a molecular switch by self-assembly on Neutravidin.
Demonstrated ability of thiazole orange dye to report nucleic acid hybridization via fluorescence.
Observed biexponential fluorescence lifetime behavior indicating intercalation and secondary binding modes.
Achieved up to a fivefold increase in fluorescence intensity upon hybridization to the target DNA.
Identified solution conditions (low pH, high ionic strength) to favor intercalative binding.

Conclusions:

The developed fluorescent molecular switch is effective for detecting nucleic acid hybridization.
Immobilization onto optical fibers creates a functional solid-phase sensor.
Modulating solution conditions optimizes dye-DNA interactions for improved sensitivity.
This platform holds promise for sensitive and regenerable nucleic acid detection systems.