Cell-penetrating poly(disulfide)s-based nanoquenchers (qCPDs) for self-monitoring of intracellular gene delivery

Monitoring gene delivery has significant benefits in gene therapy. Herein, we reported a nanoquencher system by doping a FRET pair during nucleic acid-assisted cell penetrating poly(disulfide)s (CPDs) formation. Our results...

Triarylborane Dyes as a Novel Non-Covalent and Non-Inhibitive Fluorimetric Markers for DPP III Enzyme

Novel dyes were prepared by simple “click CuAAC” attachment of a triarylborane–alkyne to the azide side chain of an amino acid yielding triarylborane dye 1 which was conjugated with pyrene (dye 2) forming a triarylborane–pyrene FRET pair. In contrast to previous cationic triarylboranes, the novel neutral dyes interact only with proteins, while their affinity to DNA/RNA is completely abolished. Both the reference triarylborane amino acid and triarylborane–pyrene conjugate bind to BSA and the hDPP III enzyme with high affinities, exhibiting a strong (up to 100-fold) fluorescence increase, whereby the triarylborane–pyrene conjugate additionally retained FRET upon binding to the protein. Furthermore, the triarylborane dyes, upon binding to the hDPP III enzyme, did not impair its enzymatic activity under a wide range of experimental conditions, thus being the first non-covalent fluorimetric markers for hDPP III, also applicable during enzymatic reactions with hDPP III substrates.

Imaging the electrical activity of organelles in living cells

Eukaryotic cells are complex systems compartmentalized in membrane-bound organelles. Visualization of organellar electrical activity in living cells requires both a suitable reporter and non-invasive imaging at high spatiotemporal resolution. Here we present hVoSorg, an optical method to monitor changes in the membrane potential of subcellular membranes. This method takes advantage of a FRET pair consisting of a membrane-bound voltage-insensitive fluorescent donor and a non-fluorescent voltage-dependent acceptor that rapidly moves across the membrane in response to changes in polarity. Compared to the currently available techniques, hVoSorg has advantages including simple and precise subcellular targeting, the ability to record from individual organelles, and the potential for optical multiplexing of organellar activity.

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay

ABSTRACTThe COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (Mpro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 Mpro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance, and characterized six FRET substrates with a 2-Abz/Tyr(3-NO2) FRET pair. The substrates 2-AbzSAVLQSGTyr(3-NO2)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and a new substrate 2-AbzVVTLQSGTyr(3-NO2)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at the position P5 improved the catalytic efficacy. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.

Visualization of lipophagy using a supramolecular FRET pair

A rationally designed supramolecular FRET pair consisting of cyanine3-cucurbit[7]uril (Cy3-CB[7]) and boron-dipyrromethene 630/650-adamantylammonium (BDP-AdA) can be used to visualize organelle-specific autophagy events. The intracellular accumulations of Cy3-CB[7] in lysosome and...

High-Resolution Single-Molecule FRET via DNA eXchange (FRET X)

ABSTRACTSingle-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object. The limited number of available FRET pair fluorophores and complicated data analysis makes it challenging to apply single-molecule FRET for structural analysis of biomolecules. Currently, only a couple of FRET pairs can be reliably measured on a single object. Here we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the FRET efficiency and pair distance with sub-nanometer resolution. We determine the distance between other pairs by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. We envision that our FRET X technology will be a tool for the high-resolution structural analysis of biomolecules and other nano-structures.

Nanoscale control of single molecule Förster resonance energy transfer by a scanning photonic nanoantenna

Förster Resonance Energy Transfer (FRET) is a widely applied technique in biology to accurately measure intra- and inter-molecular interactions at the nanometre scale. FRET is based on near-field energy transfer from an excited donor to a ground state acceptor emitter. Photonic nanoantennas have been shown to modify the rate, efficiency and extent of FRET, a process that is highly dependent on the near-field gradient of the antenna field as felt by the emitters, and thus, on their relative distance. However, most of the experiments reported to date focus on fixed antennas where the emitters are either immobilized or diffusing in solution, so that the distance between the antenna and the emitters cannot be manipulated. Here, we use scanning photonic nanoantenna probes to directly modulate the FRET efficiency between individual FRET pairs with an unprecedented nanometric lateral precision of 2 nm on the antenna position. We find that the antenna acts as an independent acceptor element, competing with the FRET pair acceptor. We directly map the competition between FRET and donor-antenna transfer as a function of the relative position between the antenna and the FRET donor-acceptor pair. The experimental data are well-described by FDTD simulations, confirming that the modulation of FRET efficiency is due to the spatially dependent coupling of the single FRET pair to the photonic antenna.