Nanopore sensing is a label-free single-molecule technique that enables the study of the dynamical structural properties of proteins. Here, we detect the translocation of cytochrome c (Cyt c) through an asymmetric thin nanopore with photothermal heating to evaluate the influence of temperature on Cyt c conformation during its translocation in an electric field. Before Cyt c translocates through an asymmetric thin SiNx nanopore, ∼1 ms trapping events occur due to electric field-induced denaturation. These trapping events were corroborated by a control analysis with a transmission electron microscopy-drilled pore and denaturant buffer. Cyt c translocation events exhibited markedly greater broad current blockade when the pores were photothermally heated. Collectively, our molecular dynamics simulation predicted that an increased temperature facilitates denaturation of the α-helical structure of Cyt c, resulting in greater blockade current during Cyt c trapping. Our photothermal heating method can be used to study the influence of temperature on protein conformation at the single-molecule level in a label-free manner.

Genomes carry the genetic blueprint of all living organisms. Their organization requires strong condensation as well as carefully regulated accessibility to specific genes for proper functioning of their hosts. The study of the structure and dynamics of the proteins that organize the genome has benefited tremendously from the development of single-molecule force spectroscopy techniques that allow for real-time, nanometer accuracy measurements of the compaction of DNA and manipulation with pico-Newton scale forces. Magnetic tweezers, in particular, have the unique ability to complement such force spectroscopy with the control over the linking number of the DNA molecule, which plays an important role when DNA-organizing proteins form or release wraps, loops, and bends in DNA. Here, we describe all the necessary steps to prepare DNA substrates for magnetic tweezers experiments, assemble flow cells, tether DNA to a magnetic bead inside a flow cell, and manipulate and record the extension of such DNA tethers. Furthermore, we explain how mechanical parameters of nucleoprotein filaments can be extracted from the data.

The binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that influence the outcome of the characterization. Tethered particle motion (TPM) is a simple, cheap, and high-throughput single-molecule method that can be used to measure binding constants of proteins binding to DNA reliably, provided that they distort DNA. In TPM, the motion of a bead tethered to a surface by DNA is tracked using light microscopy. A protein binding to the DNA will alter bead motion. This change in bead motion makes it possible to measure the DNA-binding properties of proteins. We use the bacterial protein integration host factor (IHF) and the archaeal histone HMfA as examples to show how specific binding to DNA can be measured. Moreover, we show how the end-to-end distance can provide structural insights into protein-DNA binding.

We perform laser spectroscopy at liquid helium temperatures (T = 2 K) to investigate single dibenzoterrylene (DBT) molecules doped in anthracene crystals of nanoscopic height fabricated by electrohydrodynamic dripping. Using high-resolution fluorescence excitation spectroscopy, we show that zero-phonon lines of single molecules in printed nanocrystals are nearly as narrow as the Fourier-limited transitions observed for the same guest-host system in the bulk. Moreover, the spectral instabilities are comparable to or less than one line width. By recording super-resolution images of DBT molecules and varying the polarization of the excitation beam, we determine the dimensions of the printed crystals and the orientation of the crystals\' axes. Electrohydrodynamic printing of organic nano- and microcrystals is of interest for a series of applications, where controlled positioning of quantum emitters with narrow optical transitions is desirable.

A DNA double-strand break (DSB) jeopardizes genome integrity and endangers cell viability. Actively transcribed genes are particularly detrimental if broken and need to be repressed. However, it remains elusive how fast the repression is initiated and how far it influences the neighboring genes on the chromosome. We adopt a recently developed, very fast CRISPR to generate a DSB at a specific genomic locus with precise timing, visualize transcription in live cells, and measure the RNA polymerase II (RNAPII) occupancy near the broken site. We observe that a single DSB represses the transcription of the damaged gene in minutes, which coincides with the recruitment of a damage repair protein. Transcription repression propagates bi-directionally along the chromosome from the DSB for hundreds of kilobases, and proteasome is evoked to remove RNAPII in this process. Our method builds a foundation to measure the rapid kinetic events around a single DSB and elucidate the molecular mechanism.

We present a powerful method for direct mRNA detection based on ligation-based recognition and in situ amplification, capable of single-cell imaging mRNA at single-nucleotide and single-molecule resolution. Attributed to the use of Splint R ligase that can ligate padlock probe with RNA as target template, this method can efficiently detect mRNA in the absence of reverse transcription. This method enables spatial localization and correlation analysis of gene expression in single cells, which helps us to elucidate gene function and regulatory mechanisms.

Ferritin, a spherical protein shell assembled from 24 subunits, functions as an efficient iron storage and release system through its channels. Understanding how various chemicals affect the structural behavior of ferritin is crucial for unravelling the origins of iron-related diseases in living organisms including humans. In particular, the influence of chemicals on ferritin\'s dynamics and iron release is barely explored at the single-protein level. Here, by employing optical nanotweezers using double-nanohole (DNH) structures, we examined the effect of ascorbic acid (reducing reagent) and pH on individual ferritin\'s conformational dynamics. The dynamics of ferritin increased as the concentration of ascorbic acid approached saturation. At pH 2.0, ferritin exhibited significant structural fluctuations and eventually underwent a stepwise disassembly into fragments. This work demonstrated the disassembly pathway and kinetics of a single ferritin molecule in solution. We identified four critical fragments during its disassembly pathway, which are 22-mer, 12-mer, tetramer, and dimer subunits. Moreover, we present single-molecule evidence of the cooperative disassembly of ferritin. Interrogating ferritin\'s structural change in response to different chemicals holds importance for understanding their roles in iron metabolism, hence facilitating further development of medical treatments for its associated diseases.

Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin β-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.

Vibrational quanta of melamine and its tautomer are analyzed at the single-molecule level on Cu(100) with inelastic electron tunneling spectroscopy. The on-surface tautomerization gives rise to markedly different low-energy vibrational spectra of the isomers, as evidenced by a shift in mode energies and a variation in inelastic cross sections. Spatially resolved spectroscopy reveals the maximum signal strength on an orbital nodal plane, excluding resonant inelastic tunneling as the mechanism underlying the quantum excitations. Decreasing the probe-molecule separation down to the formation of a chemical bond between the melamine amino group and the Cu apex atom of the tip leads to a quenched vibrational spectrum with different excitation energies. Density functional and electron transport calculations reproduce the experimental findings and show that the shift in the quantum energies applies to internal molecular bending modes. The simulations moreover suggest that the bond formation represents an efficient manner of tautomerizing the molecule.

Electrochemiluminescence (ECL) is rapidly evolving from an analytical method into an optical microscopy. The orthogonality of the electrochemical trigger and the optical readout distinguishes it from classic microscopy and electrochemical techniques, owing to its near-zero background, remarkable sensitivity, and absence of photobleaching and phototoxicity. In this minireview, we summarize the recent advances in ECL imaging technology, emphasizing original configurations which enable the imaging of biological entities and the improvement of the analytical properties by increasing the complexity and multiplexing of bioassays. Additionally, mapping the (electro)chemical reactivity in space provides valuable information on nanomaterials and facilitates deciphering ECL mechanisms for improving their performances in diagnostics and (electro)catalysis. Finally, we highlight the recent achievements in imaging at the ultimate limits of single molecules, single photons or single chemical reactions, and the current challenges to translate the ECL imaging advances to other fields such as material science, catalysis and biology.