title: Time-Resolved Nucleic Acid Hybridization Beacons Utilizing Unimolecular and Toehold-Mediated Strand Displacement Designs.
abstract: Nucleic acid hybridization probes are sought after for numerous assay and imaging applications. These probes are often limited by the properties of fluorescent dyes, prompting the development of new probes where dyes are paired with novel or nontraditional luminescent materials. Luminescent terbium complexes are an example of such a material, and these complexes offer several unique spectroscopic advantages. Here, we demonstrate two nonstem-loop designs for light-up nucleic acid hybridization beacons that utilize time-resolved Förster resonance energy transfer (TR-FRET) between a luminescent Lumi4-Tb cryptate (Tb) donor and a fluorescent reporter dye, where time-resolved emission from the dye provides an analytical signal. Both designs are based on probe oligonucleotides that are labeled at their opposite termini with Tb and a fluorescent reporter dye. In one design, a probe is partially blocked with a quencher dye-labeled oligonucleotide, and target hybridization is signaled through toehold-mediated strand displacement and loss of a competitive FRET pathway. In the other design, the intrinsic folding properties of an unblocked probe are utilized in combination with a temporal mechanism for signaling target hybridization. This temporal mechanism is based on a recently elucidated "sweet spot" for TR-FRET measurements and exploits distance control over FRET efficiencies to shift the Tb lifetime within or outside the time-gated detection window for measurements. Both the blocked and unblocked beacons offer nanomolar (femtomole) detection limits, response times on the order of minutes, multiplexing through the use of different reporter dyes, and detection in complex matrices such as serum and blood. The blocked beacons offer better mismatch selectivity, whereas the unblocked beacons are simpler in design. The temporal mechanism of signaling utilized with the unblocked beacons also plays a significant role with the blocked beacons and represents a new and effective strategy for developing FRET probes for bioassays.
authors.affiliation: Department of Chemistry, University of British Columbia , 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: Melissa
authors.initials: M
authors.lastname: Massey
authors.forename: Mario G
authors.initials: MG
authors.lastname: Ancona
authors.forename: Igor L
authors.initials: IL
authors.lastname: Medintz
authors.affiliation: Department of Chemistry, University of British Columbia , 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: W Russ
authors.initials: WR
authors.lastname: Algar
mesh_terms: D043844:Crown Ethers / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D049449:Luminescence; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry*; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D013725:Terbium / Q000737:chemistry; D013997:Time Factors
chemical_list: D043844:Crown Ethers; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009942:Organometallic Compounds; D013725:Terbium