PT - JOURNAL ARTICLE AU - Fengqian Chen AU - Qi Liu AU - Terrell Hilliard AU - Tingzeng Wang AU - Ziye Dong AU - Wei Li AU - Hongjun Liang AU - Weimin Gao AU - Leaf Huang AU - Degeng Wang TI - Enabling <em>in vivo</em> Analysis Via Nanoparticle-mediated Intracellular Assay Probe Delivery: Using RAS as the Prototype AID - 10.1101/2020.07.05.188862 DP - 2021 Jan 01 TA - bioRxiv PG - 2020.07.05.188862 4099 - http://biorxiv.org/content/early/2021/10/05/2020.07.05.188862.short 4100 - http://biorxiv.org/content/early/2021/10/05/2020.07.05.188862.full 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.