Molecular beacons (MBs) represent a class of nucleic acid probes with unique DNA hairpin structures that specifically target complementary DNA or RNA. The inherent “OFF” to “ON” signal transduction mechanism of MBs makes them promising molecular probes for real-time imaging of DNA/RNA in living cells. However, conventional MBs have been challenged with such issues as false-positive signals and poor biostability in complex cellular matrices. This paper describes the novel engineering steps used to improve the fluorescence signal and reduce to background fluorescence, as well as the incorporation of unnatural nucleotide bases to increase the resistance of MBs to nuclease degradation for application in such fields as chemical analysis, biotechnology, and clinical medicine. The applications of these de novo MBs for single-cell imaging will be also discussed. 1. Introduction Over the past decade, the molecular processes inside cells have been intensively investigated, including, for example, translocation of proteins and the dynamics of transcription and translation, directly affecting the fields of molecular cell biology, drug discovery, and medical diagnostics [1]. The key to the effective and successful monitoring of single-cell dynamics is the development of ultrasensitive and quantitative imaging with specific recognition of targets in living cells. To accomplish this, various nucleic acid (NA) probes, in particular, molecular beacons, have been proposed on the basis of their facile synthesis, unique functionality, molecular specificity, and structural tolerance to various modifications [2]. Since the first report of MBs in 1996 [3], they have become widely used for real-time observation of RNA distribution and dynamics in living cells. As shown in Figure 1, molecular beacons are hairpin-shaped oligonucleotides with a fluorescence donor on one end and an acceptor on the other end. Generally, molecular beacons are composed of a 15–30 base loop region for target recognition and a double-stranded stem containing 4–6 base pairs. The signal transduction mechanism of molecular beacons is mainly based on fluorescence resonance energy transfer (FRET). A fluorescence donor in the excited state transfers the absorbed energy to a nearby fluorescence acceptor via dipole-dipole coupling, causing fluorescence emission by the acceptor and/or quenching of fluorescence donor. Because the efficiency of energy transfer is significantly affected by the distance between the donor and the acceptor, the decrease in donor fluorescence and/or the increase in acceptor
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