Recently, Dr. Antony K. Chen, an assistant professor of the Department of Biomedical Engineering, College of Engineering, developed a new molecular imaging probe that enables live-cell imaging of RNAs with single-molecule sensitivity. The research was published in Biomaterials. The title of the work is “Single-Molecule Detection and Tracking of RNA Transcripts in Living Cells Using Phosphorothioate-Optimized 2’-O-Methyl RNA Molecular Beacons” http://www.sciencedirect.com/science/article/pii/S0142961216301958
Molecular beacons (MBs) are stem-loop forming, oligonucleotide-based fluorogenic probes that have the great potential for imaging specific RNAs in living cells, but their tendency to generate false-positive signals as a result of nuclease degradation and/or nonspecific binding limits sensitive and accurate intracellular RNA imaging (Fig 1A and 1B).
Figure 1. Conventional Molecular Beacons. A) Schematic representation of MBs in unhybridized and hybridized states. B) Nuclear sequestration of MBs. MBs quickly traverse to the nucleus within minutes after being injected into the cytosol of living cells.
By optimizing the MB backbone chemistry, Dr. Chen’s group developed a new chimeric MB design composed of 2’-O-methyl RNA (2Me) and a fully phosphorothioate (PS)-modified loop domain with a phosphodiester stem (2Me/PSLOOP MB). They demonstrated that this optimized MB reduces the level of false-positive signal seen compared with other PS-modified and non-PS-modified MB designs in living cells. They further showed this 2Me/PSLOOP MB could be used to accurately visualize the distribution and measure the dynamics of single mRNA transcripts in both the nucleus and the cytoplasm without interfering with gene expression or causing cytotoxicity (Fig 2A, 2B). Given the growing interest in discovering new roles and functions of RNAs in various cellular and pathological contexts, the 2Me/PSLOOP MB developed in this study could be a useful tool for imaging RNA expression, trafficking and localization in living cells with single-molecule sensitivity.
Figure 2. Detection of single RNA transcripts using 2Me/PSLOOP MBs. 2Me/PSLOOP MB and fluorescence in situ hybridization signals colocalize extensively at the single-molecule level. Figure 2B. 2Me/PSLOOP MBs measure the diffusion kinetics of single RNA transcripts in the nucleus and the cytoplasm.
Dan Zhao, Yantao Yang, Na Qu, Mingming Chen and Zhao Ma all contributed significantly to the project. Dr. Antony K. Chen is the corresponding author of the paper. The work was supported in part by the National Natural Science Foundation of China, the Beijing Natural Science Foundation and China’s 1000 Young Talent Award program.