An Elegant Biosensor Molecular Beacon Probe: Challenges and Recent Solutions

Abstract

Molecular beacon (MB) probes are fluorophore- and quencher-labeled short synthetic DNAs folded in a stem-loop shape. Since the first report by Tyagi and Kramer, it has become a widely accepted tool for nucleic acid analysis and triggered a cascade of related developments in the field of molecular sensing. The unprecedented success of MB probes stems from their ability to detect specific DNA or RNA sequences immediately after hybridization with no need to wash out the unbound probe (instantaneous format). Importantly, the hairpin structure of the probe is responsible for both the low fluorescent background and improved selectivity. Furthermore, the signal is generated in a reversible manner; thus, if the analyte is removed, the signal is reduced to the background. This paper highlights the advantages of MB probes and discusses the approaches that address the challenges in MB probe design. Variations of MB-based assays tackle the problem of stem invasion, improve SNP genotyping and signal-to-noise ratio, as well as address the challenges of detecting folded RNA and DNA.

Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors

“…Among all currently available mix-and-read fluorescent probes (Kolpashchikov 2010), including molecular beacon (MB) probes (Tyagi and Kramer 1996;Kolpashchikov 2012) and enzyme-assisted target recycling techniques (Gerasimova and Kolpashchikov 2014) such as TaqMan probe, BiDZ assay is the most promising to use on cells because of the following characteristics: (i) Unlike the MB probe (Gerasimova and Kolpashchikov 2013b), the reporter (fluorogenic substrate) produces minimal background signal when mixed with cell lysate components; (ii) it is more sensitive than the MB probe due to catalytic amplification of the fluorescent signal; and (iii) the BiDz assay does not require perishable protein enzymes, which makes it robust and cost-efficient.…”

A universal design for a DNA probe providing ratiometric fluorescence detection by generation of silver nanoclusters

“…probe, 10 µL of 10 µM target, 20 µL of 20 mM buffer, 5 µL of 0.2 mM AgNO 3 , 5 µL of 0.1 mM NaBH 4 ). For sensitivity measurements, the final concentration of AgNO 3 and NaBH 4 were scaled with concentration of probe (4 µM or 150 µM) to maintain the same concentration ratios. In the presence of 5× excess of target DNA, probe samples failed to produce a fluorescent signal, presumably due to competition between probe and target for Ag + .…”

Application and Utility of Pharmacogenetics in the Clinical Laboratory

“…Their high sensitivity is primarily due to their high signal-to-background ratio and selectivity is attributed to their hairpin confirmation requiring for complete annealing to the target in order to be dissolved [119,120]. However, even though software has been developed to assist with probe design, it is still believed to be challenging and time consuming [121].…”