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US 2020/0048710 A1Published application
Multiplexed and recyclable single-molecule sensors for quantitative analysis of nucleic-acid biomarkers
AssigneeVirginia Commonwealth University
InventorsSoma Dhakal, Anisa Kaur
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Abstract
Disclosed herein is a simple, sensitive, and fully-recyclable fluorescence resonance energy transfer (FRET)-based multiplex detection platform that overcomes current requirements of complex labeling schemes and complicated data analysis algorithms for employing single-molecule FRET (smFRET) microscopy in multiplexing. While conventional smFRET detection techniques allow for the analysis of one target at a time, the disclosed approach utilizes the gaps between high- and low-FRET signals to provide simultaneous detection and quantification of multiple nucleic acid targets.
Claims11
A hairpin-based sensor, comprising
- a first single-stranded DNA oligonucleotide (“first oligo”) having a 5′ end and a 3′ end and comprising a DNA hairpin region flanked by a 5′ flanking region and a 3′ flanking region;
- a second single-stranded DNA oligonucleotide (“second oligo”) having a 5′ end and a 3′ end and comprising a nucleic acid sequence complementary to at least a portion of the 3′ flanking region;
- a third single-stranded DNA oligonucleotide (“third oligo”) having a 5′ end and a 3′ end and comprising a nucleic acid sequence complementary to at least a portion of the 5′ flanking region;
- a fourth single-stranded DNA oligonucleotide (“probe”) having a 5′ end and a 3′ end, comprising a nucleic acid sequence complementary to at least a portion of the DNA hairpin region, and comprising a nucleic acid sequence complementary to at least a portion of a target DNA or RNA molecule;
- a first fluorescent molecule conjugated to the second oligo;
- and
- a second fluorescent molecule conjugated to the third oligo;
- wherein the first fluorescent molecule and the second fluorescent molecule together form a fluorescence resonance energy transfer (FRET) pair,
- wherein the FRET pair emit at a first-FRET efficiency when the probe is binding the DNA hairpin region, and
- wherein the FRET pair emit at a higher second-FRET efficiency when the probe binds the target DNA or RNA molecule, displacing the probe from the DNA hairpin region, allowing the DNA hairpin region to form a DNA hairpin, and shortening the distance between the first fluorescent molecule and the second fluorescent molecule.
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