Monitoring Nitric Oxide in Subcellular Compartments by Hybrid Probe Based on Rhodamine Spirolactam and SNAP-tag.

By connection of O(6)-benzylguanine (BG) to an "o-phenylenediamine-locked" rhodamine spirolactam responsive to nitric oxide (NO), a novel substrate (TMR-NO-BG) of genetically encoded SNAP-tag has been constructed. In living cells, labeling SNAP-tag fused proteins with TMR-NO-BG will in situ generate corresponding probe-protein conjugates (TMR-NO-SNAP) that not only inherit high NO sensitivity from the small-molecule parent but also guarantee the site-specificity to the designated subcellular compartments such as the mitochondrial inner membrane, nucleus, and cytoplasm. In two representative cellular processes, TMR-NO-BG demonstrates its applicability to monitor endogenous subcellular NO in the activated RAW264.7 cells stimulated by lipopolysaccharide and in the apoptotic COS-7 cells induced by etoposide.