Multiplexed and Millimeter-Scale Fluorescence Nanoscopy of Cells and Tissue Sections via Prism-Illumination and Microfluidics-Enhanced DNA-PAINT.

Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has historically afforded a small field-of-view (FOV) of around 50 μm × 50 μm at once and more recently up to ∼200 μm × 200 μm. Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates, adding cost and sample constraints to the applications. Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT (PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale FOVs.

A phosphoinositide switch mediates exocyst recruitment to multivesicular endosomes for exosome secretion.

Exosomes are secreted to the extracellular milieu when multivesicular endosomes (MVEs) dock and fuse with the plasma membrane. However, MVEs are also known to fuse with lysosomes for degradation. How MVEs are directed to the plasma membrane for exosome secretion rather than to lysosomes is unclear.

Fast and Multiplexed Super Resolution Imaging of Fixed and Immunostained Cells with DNA-PAINT-ERS.

Recent advances in super resolution microscopy have enabled imaging at the 10-20 nm scale on a light microscope, providing unprecedented details of native biological structures and processes in intact and hydrated samples. Of the existing strategies, DNA points accumulation in imaging nanoscale topography (DNA-PAINT) affords convenient multiplexing, an important feature in interrogating complex biological systems. A practical limitation of DNA-PAINT, however, has been the slow imaging speed.

Nanoscopic Spatial Association between Ras and Phosphatidylserine on the Cell Membrane Studied with Multicolor Super Resolution Microscopy.

Recent work suggests that Ras small GTPases interact with the anionic lipid phosphatidylserine (PS) in an isoform-specific manner, with direct implications for their biological functions. Studies on PS-Ras associations in cells, however, have relied on immuno-EM imaging of membrane sheets. To study their spatial relationships in intact cells, we have combined the use of Lact-C2-GFP, a biosensor for PS, with multicolor super resolution imaging based on DNA-PAINT.

Ultrafast folding kinetics of WW domains reveal how the amino acid sequence determines the speed limit to protein folding.

Protein (un)folding rates depend on the free-energy barrier separating the native and unfolded states and a prefactor term, which sets the timescale for crossing such barrier or folding speed limit. Because extricating these two factors is usually unfeasible, it has been common to assume a constant prefactor and assign all rate variability to the barrier. However, theory and simulations postulate a protein-specific prefactor that contains key mechanistic information.

Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay.

Detection of specific nucleic acid sequences is invaluable in biological studies such as genetic disease diagnostics and genome profiling. Here, we developed a highly sensitive and specific detection method that combines an advanced oligonucleotide ligation assay with multicolor single-molecule fluorescence. We demonstrated that under our experimental conditions, 7-nucleotide long DNA barcodes have the optimal short length to ascertain specificity while being long enough for sufficient ligation.

Protein Folding Cooperativity and Thermodynamic Barriers of the Simplest β-Sheet Fold: A Survey of WW Domains.

Theory and experiments have shown that microsecond folding proteins exhibit characteristic thermodynamic properties that reflect the limited cooperativity of folding over marginal barriers (downhill folding). Those studies have mostly focused on proteins with large α-helical contents and small size, which tend to be the fastest folders. A key open question is whether such properties are also present in the fastest all-β proteins.

Single-molecule peptide fingerprinting.

Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study challenging.

Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute.

Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophilus (TtAgo) can degrade double-stranded DNA (dsDNA), thereby generating small dsDNA fragments that subsequently are loaded onto TtAgo.

Single-molecule pull-down for investigating protein-nucleic acid interactions.

The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids.

A Dynamic Search Process Underlies MicroRNA Targeting.

Argonaute proteins play a central role in mediating post-transcriptional gene regulation by microRNAs (miRNAs). Argonautes use the nucleotide sequences in miRNAs as guides for identifying target messenger RNAs for repression. Here, we used single-molecule FRET to directly visualize how human Argonaute-2 (Ago2) searches for and identifies target sites in RNAs complementary to its miRNA guide.

Energy transfer processes in the isolated core antenna complexes CP43 and CP47 of photosystem II.

The energy equilibration and transfer processes in the isolated core antenna complexes CP43 and CP47 of photosystem II have been studied by steady-state and ultrafast (femto- to nanosecond) time-resolved spectroscopy at room temperature. The annihilation-free femtosecond absorption data can be described by surprisingly simple sequential kinetic models, in which the excitation energy transfer between blue and red states in both antenna complexes is dominated by sub-picosecond processes and is completed in less than 2ps. The slowest energy transfer steps with lifetimes in the range of 1-2ps are assigned to transfer steps between the chlorophyll layers located on the stromal and lumenal sides.

A photoprotection mechanism involving the D(2) branch in photosystem II cores with closed reaction centers.

Nanosecond transient absorption spectroscopy has been used to study reaction centre (RC) chlorophyll triplet quenching by carotenoid in intact photosystem II cores from T. elongatus with closed RCs. We found a triplet beta-carotene ((3)Car) signal (absorption difference maximum at 530 nm) that is sensitized by the RC chlorophyll (Chl) triplet with a formation time of ca.

The role of TyrD in the electron transfer kinetics in Photosystem II.

Redox-active tyrosine (Tyr) D is indirectly involved in controlling the primary electron transfer in PSII. The presence of the oxidized TyrD renders P680+ more oxidizing by localizing the charge more on PD1 and thus facilitates trapping of the excitation energy in PSII. We also conclude that the mechanism of the primary charge separation and stabilization is altered upon QA reduction.