RT Journal Article SR Electronic T1 Enabling in vivo Analysis Via Nanoparticle-mediated Intracellular Assay Probe Delivery: Using RAS as the Prototype JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.07.05.188862 DO 10.1101/2020.07.05.188862 A1 Fengqian Chen A1 Qi Liu A1 Terrell Hilliard A1 Tingzeng Wang A1 Ziye Dong A1 Wei Li A1 Hongjun Liang A1 Weimin Gao A1 Leaf Huang A1 Degeng Wang YR 2021 UL http://biorxiv.org/content/early/2021/10/05/2020.07.05.188862.abstract AB Many experimental protocols must be executed in vitro due to a lack of cell-permeable analysis probes. For instance, the cellular signaling moderator RAS proteins alternate between the active GTP-binding and the inactive GDP-binding states. Though many GTP analogs can serve as probes for RAS activity analysis, their cell impermeability renders in vivo analysis impossible. On the other hand, the lipid/calcium/phosphate (LCP) nanoparticle has enabled efficient intracellular delivery of a nucleotide analog as a chemotherapy agent. Thus, using RAS analysis and LCP nanoparticle as the prototype, we tackled the cell-impermeability issue via nanoparticle-mediated intracellular delivery of the analysis probe. Briefly, BODIPY-FT-GTP-γ-S, a GTP analog that becomes fluorescent only upon protein binding, was chosen as the analysis probe, so that GTP binding can be quantified by fluorescent activity. BODIPY-FT-GTP-γ-S-loaded LCP-nanoparticle was synthesized for efficient intracellular BODIPY-FT-GTP-γ-S delivery. Binding of the delivered BODIPY-FT-GTP-γ-S to the RAS proteins were consistent with previously reported observations; the RAS GTP binding activity was reduced in serum-starved cells; and a transient activation peak of the binding activity was observed upon subsequent serum reactivation of the cells. In a word, nanoparticle-mediated probe delivery enabled an in vivo RAS analysis method. The approach should be applicable to a wide variety of analysis protocols.Competing Interest StatementThe authors have declared no competing interest.