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.

Principles and Applications of Nucleic Acid Strand Displacement Reactions

“…Rather than focusing on single base mismatches, a wide variety of sensors have been described for the specific detection of nucleic acid sequences. This involves toehold-mediated strand displacement combined with molecular beacons, dendritic assemblies from DNA and PNA, and various sensors based on fluorogenic G quadruplexes. , Strand displacement schemes were combined with surface enhanced Raman scattering (SERS), electrochemiluminescence, or enzymatic amplification by acetylcholinesterase, to name but a few. Depending on the specific scheme and the number of amplification steps, limits of detection in the picomolar, femtomolar, ,,, or even attomolar range ,, were reported.…”

Lanthanide-based luminescence biolabelling

“…Several of the above mentioned studies were proof-of-concepts for multiplexed (multicolour) detection and did not contain any biomolecules. [70][71][72]76,[80][81][82] On the other hand, many multiplexed bioanalytical applications were demonstrated, which used various biomolecules, such as biotin/(strept)avidin, 73,92,93,95,96,98,103,104,107 nucleic acids, 73,75,89,93,[95][96][97]104,105,[111][112][113] peptides, 73,[88][89][90][91][92]94,95,101,102,109 This journal is © The Royal Society of Chemistry 2016 and antibodies, 73,74,78,94,95,[98][99][100][101]...…”

Time-Gated FRET and DNA-Based Photonic Molecular Logic Gates: AND, OR, NAND, and NOR

“…The protective and highly stable environment that the Lumi4 cryptand provides for the Tb­(III) lends exceptional brightness to this LLC. Methods for labeling oligonucleotides with A546 or A488 maleimide and Lumi4-Tb-NHS are described in the SI …”

“…Methods for labeling oligonucleotides with A546 or A488 maleimide and Lumi4-Tb-NHS are described in the SI. 43 Absorbance and PL Measurements. An Infinite M1000 Pro multifunction plate reader (Tecan, Morrisville, NC) was used for absorbance and PL measurements and offered monochromator-based selection of excitation/emission wavelengths, as well as lag time and detector integration time settings for time-gated measurements.…”