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Genetically encoded light-up RNA aptamers have become powerful tools for visualizing RNAs in cells. However, fluorogenic dye/aptamer pairs used for super-resolution RNA imaging are still rare. Herein, we reported an environment-sensitive dye Nap which can light up its target RNA aptamer NapRA for aptamer-based RNA imaging with confocal and structured illumination microscopy (SIM). Nap was designed to be amphiphilic to assemble into nano-particles in water associated with the quenched fluorescence by the aggregation effects. NapRA was obtained from SELEX technology with a 12-round Nap -based selection and has a high binding affinity to Nap of K d = 3.142 nM. The super affinity disintegrated the dye aggregate and resulted in a turn-on fluorescent signal. The light-up feature combined with high photostability made Nap / NapRA a useful system capable of super-resolution imaging of the expression of RNA in bacteria. This work represents the first application of dye aggregation-disaggregation for aptamer-based RNA imaging. [Display omitted] • A novel fluorogenic dye-aptamer pair was developed for RNA imaging. • Aggregation-disaggregation property of dye Nap was used to light up aptamer. • Aptamers targeting Nap of K d = 3.142 nM was screened by SELEX. • Super-resolution imaging of bacteria mRNA was achieved. [ABSTRACT FROM AUTHOR]

The visualization of self‐assembled structure and dynamics at the molecular level has become a powerful method to understand structure–function relationships of self‐assembly. Herein, we in situ real‐time imaged the dynamic process of benzyl‐naphthalimide dyes at the nanoscale and inspected their internal structure with minimum 2.8 nm localization accuracy through single‐molecule localization microscopy (SMLM) imaging. We monitored the growth process of three different assemblies in situ, which possessed highly heterogeneous dynamics with different shapes and growth rates. Furthermore, diverse growth rates were also found at different sites in the same assembly. These results highlight the application of super‐resolution microscopy techniques for real‐time visualization of internal assembled structure and dynamics in situ. [ABSTRACT FROM AUTHOR]

The visualization of self‐assembled structure and dynamics at the molecular level has become a powerful method to understand structure–function relationships of self‐assembly. Herein, we in situ real‐time imaged the dynamic process of benzyl‐naphthalimide dyes at the nanoscale and inspected their internal structure with minimum 2.8 nm localization accuracy through single‐molecule localization microscopy (SMLM) imaging. We monitored the growth process of three different assemblies in situ, which possessed highly heterogeneous dynamics with different shapes and growth rates. Furthermore, diverse growth rates were also found at different sites in the same assembly. These results highlight the application of super‐resolution microscopy techniques for real‐time visualization of internal assembled structure and dynamics in situ. [ABSTRACT FROM AUTHOR]

Super-resolution fluorescence microscopy has emerged as a powerful tool for studying mitochondrial dynamics in living cells. However, the lack of photostable and chemstable probe makes long-term super-resolution imaging of mitochondria still a challenging work. Herein, we reported a 4-azetidinyl-naphthliamide derived SNAP-tag probe AN-BG exhibiting excellent fluorogenicity and photostability for long-term super-resolution imaging of mitochondrial dynamics. The azetidinyl group and naphthalimide fluorophore are in a flat conformation which can effectively suppress twisted intramolecular charge transfer and then effectively improve the brightness and photostability. This planarized molecular structure is conducive to the formation of fluorescence-quenched J -aggregates, and the protein labeling process will depolymerize the probes and restore fluorescence. Fluorescent labeling mitochondrial inner membrane proteins via SNAP tags overcomes the shortcomings that variations in mitochondrial inner membrane potential will release probes attached to mitochondria by electrostatic interactions. Therefore, AN-BG realized the stable labeling of mitochondria and the long-term imaging of mitochondrial dynamics under super-resolution microscopy. Image 1 • A naphthliamide-derived fluorogenic probe for SNAP-tag was developed. • Fluorescent labeling of mitochondria was achieved via SNAP-tag fused proteins. • Long-term super-resolution imaging of mitochondrial dynamics was achieved. • The dynamic mitochondrial fusion and fission were imaged with fluorescent nanoscopy. • Two-color SIM imaging revealed multiple modes of action between mitochondria and lysosomes. [ABSTRACT FROM AUTHOR]