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bioRxiv · 10.1101/2025.03.22.644677

NitrOFF: An engineered fluorescent biosensor to illuminate nitrate transport in living cells

Cook, M.·Smailys, J.·Ji, K.·Phelps, S.·Tutol, J.·Kim, W.·Ong, W.·Peng, W.·Maydew, C.·Zhang, Y.·Dodani, S. C.
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Abstract

The duality of nitrate is nowhere best exemplified than in human physiology - a detrimental pollutant but also a protective nutrient and signaling ion - particularly as connected to reactive nitrogen oxides. Aside from limited insights into nitrate uptake and storage, foundational nitrate biology has lagged. Genetically encoded fluorescent biosensors can address this gap with real-time imaging. However, imaging technologies for mammalian cell applications remain rare. Here, we set out to design and engineer a two-domain chimera fusing the split green fluorescent protein EGFP and the nitrate recognition domain NreA from Staphylococcus carnosus. Over 7 rounds of directed evolution, 15 mutations were accumulated resulting in the functional biosensor NitrOFF. NitrOFF has a high degree of allosteric communication between the domains reflected in a turn-off intensiometric response (Kd {approx} 9 {micro}M). This was further reinforced by X-ray crystal structures of apo and nitrate bound NitrOFF, which revealed that the two domains undergo a large-scale conformational rearrangement that changes the relative positioning of the EGFP and NreA domains by 68.4{degrees}. Such a dramatic difference was triggered by the formation of a long helix at the engineered linker connecting the two domains, peeling the {beta}7 strand off the EGFP and thus extinguishing the fluorescence upon nitrate binding. Finally, as a proof-of-concept, we highlighted the utility of this first-generation biosensor to monitor exogenous nitrate uptake and modulation in a human embryonic kidney (HEK) 293 cell line. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/644677v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1ba853eorg.highwire.dtl.DTLVardef@81bed9org.highwire.dtl.DTLVardef@18a313org.highwire.dtl.DTLVardef@17a5e80_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Cook, M., Smailys, J., Ji, K., Phelps, S., Tutol, J., Kim, W., Ong, W., Peng, W., Maydew, C., Zhang, Y., Dodani, S. C.. 2025-03-23. NitrOFF: An engineered fluorescent biosensor to illuminate nitrate transport in living cells. https://doi.org/10.1101/2025.03.22.644677

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