Recent Advances in the Molecular Beacon Technology for Live-Cell Single-Molecule Imaging

Abstract

Summary Nucleic acids, aside from being best known as the carrier of genetic information, are versatile biomaterials for constructing nanoscopic devices for biointerfacing, owing to their unique properties such as specific base pairing and predictable structure. For live-cell analysis of native RNA transcripts, the most widely used nucleic acid-based nanodevice has been the molecular beacon (MB), a class of stem-loop-forming probes that is activated to fluoresce upon hybridization with target RNA. Here, we overview efforts that have been made in developing MB-based bioassays for sensitive intracellular analysis, particularly at the single-molecule level. We also describe challenges that are currently limiting the widespread use of MBs and provide possible solutions. With continued refinement of MBs in terms of labeling specificity and detection accuracy, accompanied by new development in imaging platforms with unprecedented sensitivity, the application of MBs is envisioned to expand in various biological research fields.

Development of a Universal RNA Dual-Terminal Labeling Method for Sensing RNA-Ligand Interactions

“…On the other hand, the dual-terminal labeling strategy should be useful for large-scale and facile production of any RNA beacon in principle. RNA beacons can be used for miRNA detection, visualization of microRNA maturation, specific RNA imaging, separation of cell populations, and identification of inhibitors of RNA-binding protein [43][44][45][46][47][48] . Therefore, it is anticipated that the RNA dual-labeling method would have widespread applications for preparation of functionalized RNA beacons.…”

Imaging of Hepatitis B Virus Nucleic Acids: Current Advances and Challenges

“…Another method commonly used for labelling of intracellular RNA are molecular beacons (MB) [ 57 ]. These nanodevices contain a target sequence flanked by self-complimentary palindromic sequences which form a stem-loop ( Figure 7 A) [ 57 , 58 ]. Typically, the 3′ and 5′ ends of the MB are conjugated with a fluorophore and quencher, respectively.…”

“…Upon hybridisation with the target sequence, the fluorophore and quencher are distanced from each other, resulting in fluorescence emission ( Figure 7 B). An advantage over aptamer-based labelling is that MBs do not require the engineering of the virus and generally exhibit low noise [ 58 ], although imperfect quenching of the fluorophore or degradation by nucleases may still result in background fluorescence [ 57 ]. Furthermore, MBs are prone to non-specific protein binding, which can lead to non-specific signals.…”

“…These limitations can be overcome by proper experimental conditions and the synthesis of MB backbones with unnatural nucleotide analogues which provide resistance to nucleases. [ 57 ]. Although MBs have shown their potential in the tracking of viral RNAs in living cells (see below), the delivery of MBs into living cells requires additional manipulations, which was achieved through methods such as microinjection and reversible permeabilization with streptolysin O [ 57 , 58 ].…”

“…[ 57 ]. Although MBs have shown their potential in the tracking of viral RNAs in living cells (see below), the delivery of MBs into living cells requires additional manipulations, which was achieved through methods such as microinjection and reversible permeabilization with streptolysin O [ 57 , 58 ].…”