![]() Strand displacement probe with a fluorophore on one strand and a quencher on the other strand was introduced by Morrison et al. The significance of MB probe can be better understood in the context of preceding instantaneous probes such as strand displacement probes (Figure 1(b)) and adjacent hybridization probes (Figure 1(c)). Therefore, here we use the terms “instantaneous format” and “instantaneous probes” to define the property of MB probes to be used without washing steps. MB probes have broader spectrum of applications and greater significance than just rtPCR. This property of a probe sometimes is referred to as “real time.” However, this term is traditionally associated with real-time PCR (rtPCR) that also uses SYBR Green, and TaqMan assay, which significantly differ from MB probe and other immediate or instant mix-and-read type of assays. (i) The probe produces a signal that enables the detection of the target immediately in homogeneous solution without the need for separation of the probe-analyte hybrid from the excess amount of the unbound probe. The most important features of MB probe include (i) s generation of fluorescent signal that can be registered immediately after hybridization event (ii) conformational constraint in the form of a stem loop (iii) reversible binding to the analyte (Figure 1(a)) as detailed below. The hybridization efficiency is assessed as a difference in donor and acceptor fluorescence. Hybridization of two fluorophore-conjugated DNA probes to the adjacent positions of an analyte results in Förster resonance energy transfer (FRET). The quencher-conjugated strand is displaced from the complex with the fluorophore-conjugated DNA by the analyte. Some important features of the probe are (i) the ability to produce instantaneous fluorescent signal (ii) conformational constraint in the form of a stem loop in the target-unbound state (left) (iii) reversibility of hybridization. (a) Classical design of molecular beacon (MB) probe. Therefore, MB probes have forestalled the rise of DNA nanomotors and nanorobots, a field that has recently received a substantial attention. The behavior of this molecule can be considered as an elementary molecular device that switches between the two conformations in an analyte-dependent manner. The emitted light can be quantified directly in the sample. ![]() Formation of the probe-analyte duplex separates the fluorophore from the quencher, thus brightening the MB’s fluorescence. ![]() In the absence of a complementary nucleic acid target (analyte), the fluorescence of the fluorophore is quenched by the closely located quencher dye. In its classical design, a molecular beacon (MB) probe is a stem-loop-folded oligodeoxyribonucleotide with fluorophore and quencher dyes conjugated to the opposite ends of the hairpin (Figure 1(a)). Introduction: An Elegant Unimolecular Biosensor 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. This paper highlights the advantages of MB probes and discusses the approaches that address the challenges in MB probe design. Furthermore, the signal is generated in a reversible manner thus, if the analyte is removed, the signal is reduced to the background. Importantly, the hairpin structure of the probe is responsible for both the low fluorescent background and improved selectivity. 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). 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. ![]() Molecular beacon (MB) probes are fluorophore- and quencher-labeled short synthetic DNAs folded in a stem-loop shape. ![]()
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