Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis

DNA-based fluorescent light-up aptamers (FLAPs) are promising for bioanalytical assays because they provide a low-cost fluorescent signal readout without the need for labeling of nucleic acid signal reporters with fluorophores and/or quenchers, unlike conventional hybridization probes used for instantaneous nucleic acid detection. Instead, FLAPs non-covalently bind dye ligands , which exhibit intrinsically low fluorescence in aqueous solutions, but become highly emitting upon FLAP binding. This protocol describes an algorithm to design split light-up aptamer sensors (SLASs) utilizing DAP-10-42, the most efficient DNA FLAP reported thus far. When equipped with nucleic acid sequences complementary to a nucleic acid target of interest, SLAS is a promising tool for nucleic acid analysis allowing for the sequence-specific detection of nucleic acid targets with selectivity down to one nucleotide to enable analysis of single-nucleotide substitutions (SNSs). SLASs offer the advantage of a label-free fluorescence-based signal readout, which can be measured with a conventional cuvette-based fluorescent spectrophotometer, a portable fluorometer, or visually observed upon excitation with a handheld light source. The SLAS approach is beneficial for biosensing applications in disease diagnostics, environmental monitoring, and biomolecular research.