Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. This is particularly challenging for measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this, we developed Corn, a genetically encoded fluorescent RNA reporter suitable for quantifying RNA transcription in cells. Corn binds and induces fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, which resembles the fluorophore found in red fluorescent protein (RFP). Notably, Corn shows high photostability, enabling quantitative fluorescence imaging of mTOR-dependent Pol III transcription. Unlike actinomycin D, we found that mTOR inhibitors resulted in heterogeneous transcription suppression in individual cells. Quantitative imaging of Corn-tagged Pol III transcript levels revealed distinct Pol III transcription “trajectories” elicited by mTOR inhibition. Together, these studies provide an approach for quantitative measurements of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA-fluorophore complex.
Oligonucleotides such as short, double-stranded RNA (siRNA) or plasmid DNA (pDNA) promise high potential in gene therapy. For pharmaceutical application, however, adequate drug carriers are required. Among various concepts progressing in the market or final development, nanosized hydrogel particles may serve as novel transport media especially for siRNA. In this work, a new concept of synthesizing polymeric cationic nanohydrogels was developed, which offers a promising strategy to complex and transport siRNA into cells. For this purpose, amphiphilic reactive ester block copolymers were synthesized by RAFT polymerization of pentafluorophenyl methacrylate as reactive ester monomer together with tri(ethylene glycol)methyl ether methacrylate. In polar aprotic solvents, a self-assembly of these polymers could be observed leading to the formation of nanometer-sized polymer aggregates. The resulting superstructures were used to convert the reactive precursor block copolymers with amine-containing cross-linker molecules into covalently stabilized hydrogel particles. Detailed dynamic light scattering studies showed that the structure of the self-assembled aggregates can permanently be locked-in by this process. This method offers a new possibility to synthesize precise nanohydrogels of different size starting from various block copolymers. Moreover, via reactive ester approach, further functionalities could be attached to the nanoparticle, such as fluorescent dyes, which allowed distinct tracing of the hydrogels during complexation with siRNA or cell uptake experiments. In this respect, cellular uptake of the particles themselves as well as with its payload could be detected successfully. Looking ahead, these novel cationic nanohydrogel particles may serve as a new platform for proper siRNA delivery systems.
Intracellular distribution of siRNA after in vitro transfection typically depends on lipopolyplexes, which must release the siRNA into the cytosol. Here, the fate of siRNAs was monitored by FRET-based live cell imaging. Subsequent to in situ observation of uptake and release processes, this approach allowed the observation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, followed, in some cases, by fast siRNA influx into the nucleus. The continued observation of intact siRNA against a background of free fluorophores resulting from advanced degradation was possible by a specifically developed imaging algorithm, which identified populations of intact siRNA in pixels based on FRET. This proved to be essential in the end point definition of siRNA distribution, which typically featured partially degraded siRNA pools in perinuclear structures. Our results depict the initial 4 h as a critical time window, characterized by fast initial burst release into the cytosol, which lay the foundations for subsequent intracellular distribution of siRNA. Combination with a subsequent slower, but sustained release from endosomal reservoirs may contribute to the efficiency and duration of RNAi, and explain the success of lipopolyplexes in RNAi experiments in cell culture.
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