Browsing by Subject "FRET"

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  • Matlashov, Mikhail E.; Vera, Jorge; Kasatkina, Ludmila A.; Khodakhah, Kamran; Verkhusha, Vladislav V. (2022)
    Near-infrared (NIR) genetically encoded calcium indicators (GECIs) are becoming powerful tools for neuroscience. Because of their spectral characteristics, the use of NIR GECIs helps to avoid signal loss from the absorption by body pigments, light-scattering, and autofluorescence in mammalian tissues. In addition, NIR GECIs do not suffer from cross-excitation artifacts when used with common fluorescent indicators and optogenetics actuators. Although several NIR GECIs have been developed, there is no NIR GECI currently available that would combine the high brightness in cells and photostability with small size and fast response kinetics. Here, we report a small FRET-based NIR fluorescent calcium indicator iGECInano. We characterize iGECInano in vitro, in non-neuronal mammalian cells, and primary mouse neurons. iGECInano demonstrates the improvement in the signal-to-noise ratio and response kinetics compared to other NIR GECIs.
  • Wang, Linping; Pogue, Sylvain; Laamanen, Karoliina; Saarela, Jani; Poso, Antti; Laitinen, Tuomo; Valkonen, Jari P. T. (2021)
    Sweet potato virus disease (SPVD), caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV), is responsible for substantial yield losses all over the world. However, there are currently no approved treatments for this severe disease. The crucial role played by RNase III of SPCSV (CSR3) as an RNA silencing suppressor during the viruses' synergistic interaction in sweetpotato makes it an ideal drug target for developing antiviral treatment. In this study, high-throughput screening (HTS) of small molecular libraries targeting CSR3 was initiated by a virtual screen using Glide docking, allowing the selection of 6,400 compounds out of 136,353. We subsequently developed and carried out kinetic-based HTS using fluorescence resonance energy transfer technology, which isolated 112 compounds. These compounds were validated with dose-response assays including kinetic-based HTS and binding affinity assays using surface plasmon resonance and microscale thermophoresis. Finally, the interference of the selected compounds with viral accumulation was verified in planta. In summary, we identified five compounds belonging to two structural classes that inhibited CSR3 activity and reduced viral accumulation in plants. These results provide the foundation for developing antiviral agents targeting CSR3 to provide new strategies for controlling sweetpotato virus diseases. IMPORTANCE We report here a high-throughput inhibitor identification method that targets a severe sweetpotato virus disease caused by coinfection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield losses. Specifically, we targeted the RNase III enzyme encoded by SPCSV, which plays an important role in suppressing the RNA silencing defense system of sweetpotato plants. Based on virtual screening, laboratory assays, and confirmation in planta, we identified five compounds that could be used to develop antiviral drugs to combat the most severe sweetpotato virus disease.
  • Koskinen, Mikko; Hotulainen, Pirta (2014)
  • Li, Lei; Hsu, Hsun-Chia; Verkhusha, Vladislav V.; Wang, Lihong V.; Shcherbakova, Daria M. (2021)
    Photoacoustic tomography (PAT) with genetically encoded near-infrared probes enables visualization of specific cell populations in vivo at high resolution deeply in biological tissues. However, because of a lack of proper probes, PAT of cellular dynamics remains unexplored. Here, the authors report a near-infrared Forster resonance energy transfer (FRET) biosensor based on a miRFP670-iRFP720 pair of the near-infrared fluorescent proteins, which enables dynamic functional imaging of active biological processes in deep tissues. By photoacoustically detecting the changes in the optical absorption of the miRFP670 FRET-donor, they monitored cell apoptosis in deep tissue at high spatiotemporal resolution using PAT. Specifically, they detected apoptosis in single cells at a resolution of approximate to 3 mu m in a mouse ear tumor, and in deep brain tumors (>3 mm beneath the scalp) of living mice at a spatial resolution of approximate to 150 mu m with a 20 Hz frame rate. These results open the way for high-resolution photoacoustic imaging of dynamic biological processes in deep tissues using NIR biosensors and PAT.