Markina N.M., Palkina K.A., Sarkisyan K.S. and Yampolsky I.V.
Planta LLC, Russia Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Russia
Posters & Accepted Abstracts: Arch Med
The fungus Neonothopanus nambi is the first eukaryotic bioluminescent organism with fully discovered enzymes, involved in luciferin biosynthesis. Also the luciferase and structure of the fungal luciferin molecule are both identified as well. Bioluminescent genetically encodable sensors that rely on BRET, bioluminescence resonance energy transfer, are widely used in biomedical research and are typically composed of luciferase and a fluorescent protein that serves as a light acceptor. The efficiency of BRET and the dynamic range of a sensor are largely determined by the spectral match between luciferase and a fluorescent protein, as well as by the structure and the length of amino acid linker that separates them. Water solubility, small size and membrane permeability of the luciferin (e.g., fungal luciferin) can also broaden the range of applications of a particular BRET system. In this study, we explored the potential of the recently discovered Neonothopanus nambi luciferase (nnLuz) for BRET-based application by analyzing the efficiency of energy transfer to several red fluorescent proteins. We assembled16 constructs containing various combinations of nnLuz, acceptor proteins TagRFP, tdTomato or mRuby2, and 8 different linkers and analyzed light emission by spectrally resolved imaging of transiently transfected HEK293T cells. This allowed us to determine promising acceptors and linkers for further construction of BRET-sensors based on fungal luciferase. The study was funded by the Ministry of Science and Higher Education of the Russian Federation, project identifier RFMEFI61317X0062.
1. Schultz DT, Kotlobay AA, Ziganshin R, Bannikov A, Markina NM, Chepurnyh TV, Shakhova ES, Palkina K, Haddock SHD, Yampolsky IV, Oba Y (2018).Corrigendum to “Luciferase of the Japanese syllid polychaete Odontosyllis undecimdonta” [Biochem. Biophys. Res. Commun. 2018 Jul 20; 502(3):318–323]. Biochem Biophys Res Commun 503 (2), 1179
2. Schultz DT, Kotlobay AA, Ziganshin R, Bannikov A, Markina NM, Chepurnyh TV, Shakhova ES, Palkina K, Haddock SHD, Yampolsky IV, Oba Y (2018).Luciferase of the Japanese syllid polychaete Odontosyllis umdecimdonta. Biochem Biophys Res Commun 502 (3), 318–323
3. Markina NM, Pereverzev AP, Staroverov DB, Lukyanov KA, Gurskaya NG (2018). Generation of cell lines stably expressing a fluorescent reporter of nonsense-mediated mRNA decay activity. Methods Mol Biol 1720, 187–204
4. Povarova NV, Markina NM, Baranov MS, Barinov NA, Klinov DV, Kozhemyako VB, Lukyanov KA (2017). A water-soluble precursor for efficient silica polymerization by silicateins. Biochem Biophys Res Commun 495 (2), 2066–2070
5. Gurskaya NG, Pereverzev AP, Staroverov DB, Markina NM, Lukyanov KA (2016). Analysis of Nonsense-Mediated mRNA Decay at the Single-Cell Level Using Two Fluorescent Proteins. Methods Enzymol 572, 291–314