RNA Probes for Visualization of Sarcin/ricin Loop Depurination without Background Fluorescence.

Protein synthesis via ribosomes is a fundamental process in all known living organisms. However, it can be completely stalled by removing a single nucleobase (depurination) at the sarcin/ricin loop of the ribosomal RNA. In this work, we describe the preparation and optimization process of a fluorescent probe that can be used to visualize depurination. Starting from a fluorescent thiophene nucleobase analog, various RNA probes that fluoresce exclusively in the presence of a depurinated sarcin/ricin-loop RNA were designed and characterized. The main challenge in this process was to obtain a high fluorescence signal in the hybridized state with an abasic RNA strand, while keeping the background fluorescence low. With our new RNA probes, the fluorescence intensity and lifetime can be used for efficient monitoring of depurinated RNA.

Inactivation of Competitive Decay Channels Leads to Enhanced Coumarin Photochemistry.

In the development of photolabile protecting groups, it is of high interest to selectively modify photochemical properties with structural changes as simple as possible. In this work, knowledge of fluorophore optimization was adopted and used to design new coumarin- based photocages. Photolysis efficiency was selectively modulated by inactivating competitive decay channels, such as twisted intramolecular charge transfer (TICT) or hydrogen-bonding, and the photolytic release of the neurotransmitter serotonin was demonstrated. Structural modifications inspired by the fluorophore ATTO 390 led to a significant increase in the uncaging cross section that can be further improved by the simple addition of a double bond. Ultrafast transient absorption spectroscopy gave insights into the underlying solvent-dependent photophysical dynamics. The chromophores presented here are excellently suited as new photocages in the visible wavelength range due to their simple synthesis and their superior photochemical properties.

Visible light-activatable Q-dye molecular beacons for long-term mRNA monitoring in neurons.

Herein, we present a new class of Q-dye molecular beacons (MBs) that can be locally activated with visible light in hippocampal neurons. Our novel architecture increases the available monitoring time for neuronal mRNA from several minutes to 14 hours, since a lower light-sampling rate is required for tracking.

Differential regulation of local mRNA dynamics and translation following long-term potentiation and depression.

Decades of work have demonstrated that messenger RNAs (mRNAs) are localized and translated within neuronal dendrites and axons to provide proteins for remodeling and maintaining growth cones or synapses. It remains unknown, however, whether specific forms of plasticity differentially regulate the dynamics and translation of individual mRNA species. To address this, we targeted three individual synaptically localized mRNAs, CamkIIa, ß-actin, Psd95, and used molecular beacons to track endogenous mRNA movements. We used reporters and CRISPR/Cas9 gene editing to track mRNA translation in cultured neurons. We found alterations in mRNA dynamic properties occurred during two forms of synaptic plasticity, long-term potentiation (cLTP) and depression (mGluR-LTD). Changes in mRNA dynamics following either form of plasticity resulted in an enrichment of mRNA in the vicinity of dendritic spines. Both the reporters and tagging of endogenous proteins revealed the transcript-specific stimulation of protein synthesis following cLTP or mGluR-LTD. As such, the plasticity-induced enrichment of mRNA near synapses could be uncoupled from its translational status. The enrichment of mRNA in the proximity of spines allows for localized signaling pathways to decode plasticity milieus and stimulate a specific translational profile, resulting in a customized remodeling of the synaptic proteome.

Photo-tethered molecular beacons for superior light-induction.

We developed a superior class of light-activatable molecular beacons with photo-tethered loop regions. Two simple modifications and probe cyclisation prevent the molecular beacon from hybridising with the target RNA before light-activation. Full activity of the molecular beacon is elicited upon illumination with 365 nm light.

Sensitized Two-Photon Activation of Coumarin Photocages.

Here we report the design of a new coumarin-based photolabile protecting group with enhanced two-photon absorption. Two-photon excited fluorescence (TPEF), color-tuned ultrafast transient absorption spectroscopy and infrared (IR) measurements are employed to photochemically characterize the newly designed ATTO 390-DEACM-cargo triad. Increased two-photon cross-section values of the novel cage in comparison to the widely used protecting group DEACM ([7-(diethylamino)coumarin-4-yl]methyl) are extracted from TPEF experiments. Femtosecond pump-probe experiments reveal a fast intramolecular charge transfer, a finding that is confirmed by quantum chemical calculations. Uncaging of glutamate is monitored in IR measurements by photodecarboxylation of the carbamate linker between the photolabile protecting group and the glutamate, showing the full functionality of the novel two-photon activatable photocage.