Towards a fluorescent molecular switch for nucleic acid biosensing |
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Authors: | Melissa Massey Ulrich J Krull |
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Institution: | (1) Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, Ontario, L5L 1C6, Canada; |
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Abstract: | A novel fluorescent molecular switch for the detection of nucleic acid hybridization has been explored in relation to the
development of a structure that would be amenable for operation when immobilized for solid-phase analyses. The structure was
prepared by self-assembly, and used Neutravidin as the central multivalent docking molecule, a newly synthesized biotinylated
long-chain linker for intercalating dye that was modified with thiazole orange (TO) at one end, and a biotinylated probe oligonucleotide.
Self-assembly of the biotinylated components on adjacent Neutravidin binding sites allowed for physical placement of an oligonucleotide
probe molecule next to tethered TO. The TO located at the end of the flexible linker chain was available to intercalate, and
could report if a duplex structure was formed by a probe–target interaction by means of fluorescence intensity. Subsequently,
regeneration of the single-stranded probe was possible without loss of the intercalator to solution. The switch constructs
were assembled in solution and subsequently immobilized onto biotin functionalized optical fibers to complete the sensor design.
Solution-phase fluorescence lifetime data showed a biexponential behavior for switch constructs, suggesting intercalation
as well as a significant secondary binding mode for the immobilized TO. It was found that the secondary binding mechanism
for the dye to DNA could be decreased, thus shifting the dye to intercalative binding modes, by adjusting the solution conditions
to a pH below the pI of Neutravidin, and by increasing the ionic strength of the buffer. Preliminary work demonstrated that
it was possible to achieve up to a fivefold increase in fluorescence intensity on hybridization to the target. |
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