Dynamics of G-Quadruplex Formation under Molecular Crowding.

G-quadruplex (G4) structures assemble from guanine-rich DNA sequences and are believed to regulate several key cellular processes. G4 formation and conformational interconversions are well-established to occur dynamically . However, a clear understanding of G4 formation dynamics in cells as well as under conditions mimicking the cellular environment is missing.

Backbone resonance assignments of the C-terminal region of human translation initiation factor eIF4B.

Translation initiation in eukaryotes is an early step in protein synthesis, requiring multiple factors to recruit the ribosomal small subunit to the mRNA 5' untranslated region. One such protein factor is the eukaryotic translation initiation factor 4B (eIF4B), which increases the activity of the eIF4A RNA helicase, and is linked to cell survival and proliferation. We report here the protein backbone chemical shift assignments corresponding to the C-terminal 279 residues of human eIF4B.

Ligand Binding to Dynamically Populated G-Quadruplex DNA.

Several small-molecule ligands specifically bind and stabilize G-quadruplex (G4) nucleic acid structures, which are considered to be promising therapeutic targets. G4s are polymorphic structures of varying stability, and their formation is dynamic. Here, we investigate the mechanisms of ligand binding to dynamically populated human telomere G4 DNA by using the bisquinolinium based ligand Phen-DC3 and a combination of single-molecule FRET microscopy, ensemble FRET and CD spectroscopies.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes.

Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions.

The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions.

A direct view of the complex multi-pathway folding of telomeric G-quadruplexes.

G-quadruplexes (G4s) are DNA secondary structures that are capable of forming and function in vivo The propensity of G4s to exhibit extreme polymorphism and complex dynamics is likely to influence their cellular function, yet a clear microscopic picture of their folding process is lacking. Here we employed single-molecule FRET microscopy to obtain a direct view of the folding and underlying conformational dynamics of G4s formed by the human telomeric sequence in potassium containing solutions. Our experiments allowed detecting several folded states that are populated in the course of G4 folding and determining their folding energetics and timescales.

Folding dynamics and conformational heterogeneity of human telomeric G-quadruplex structures in Na+ solutions by single molecule FRET microscopy.

G-quadruplex structures can occur throughout the genome, including at telomeres. They are involved in cellular regulation and are potential drug targets. Human telomeric G-quadruplex structures can fold into a number of different conformations and show large conformational diversity.

Dynamics of fluorescent dyes attached to G-quadruplex DNA and their effect on FRET experiments.

FRET spectroscopy is a promising approach for investigating the dynamics of G-quadruplex DNA folds and improving the targeting of G-quadruplexes by potential anticancer compounds. To better interpret such experiments, classical and replica-exchange molecular dynamics simulations and fluorescence-lifetime measurements are used to understand the behavior of a range of Cy3-based dyes attached to the 3' end of G-quadruplex DNA. The simulations revealed that the dyes interact extensively with the G-quadruplex.

Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells.

Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro. Their application in vivo, however, has remained challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Förster resonance energy transfer (FRET) spectroscopy.

Pure Mitochondrial DNA Does Not Activate Human Neutrophils in vitro.

Excessive activation of the innate immune system often leads to fatal consequences and can be considered as one of the phenoptotic events. After traumatic injury, various components of mitochondria are released into the circulation and stimulate myeloid cells of the innate immunity. Presumably, mitochondrial DNA (mtDNA) might activate immune cells (Zhang, Q.

Single-molecule spectroscopy of cold denaturation and the temperature-induced collapse of unfolded proteins.

Recent Förster resonance energy transfer (FRET) experiments show that heat-unfolded states of proteins become more compact with increasing temperature. At the same time, NMR results indicate that cold-denatured proteins are more expanded than heat-denatured proteins. To clarify the connection between these observations, we investigated the unfolded state of yeast frataxin, whose cold denaturation occurs at temperatures above 273 K, with single-molecule FRET.

Study on the interaction between isoniazid and bovine serum albumin by fluorescence spectroscopy: the effect of dimethylsulfoxide.

The investigation of the binding between isoniazid (or isonicotinic acid hydrazide, INH) and serum albumin is of crucial importance to reveal the reason of resistant Mycobacterium tuberculosis strains towards INH and to increase the anti-tuberculous activity of INH. The interaction between INH and bovine serum albumin (BSA) was studied by fluorescence, UV and FT-IR spectroscopy methods. The analysis of the emission quenching at different temperatures revealed that the quenching mechanism corresponds to a static process and, as consequence; a complex INH-BSA is formed.