Observing G4 formation and its resolution by Pif1 in real time by manipulation under magnetic tweezers.

G-quadruplexes (G4s) are nucleic acids secondary structures that may form in guanine-rich sequences, either intra or inter-molecularly. Ability of a primary sequence to form a G4 can be predicted computationally with an improving accuracy as well as tested in bulk using biophysical measurements. As a result, G4 density maps have been devised for a large number of genomes from all life kingdoms.

Magnetic tweezers principles and promises.

Magnetic tweezers have become popular with the outbreak of single molecule micromanipulation: catching a single molecule of DNA, RNA or a single protein and applying mechanical constrains using micron-size magnetic beads and magnets turn out to be easy. Various factors have made this possible: the fact that manufacturers have been preparing these beads to catch various biological entities-the ease of use provided by magnets which apply a force or a torque at a distance thus inside a flow cell-some chance: since the forces so generated are in the right range to stretch a single molecule. This is a little less true for torque.

Fluorescence to measure light intensity.

Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied.

Displacement and dissociation of oligonucleotides during DNA hairpin closure under strain.

The hybridization kinetic of an oligonucleotide to its template is a fundamental step in many biological processes such as replication arrest, CRISPR recognition, DNA sequencing, DNA origami, etc. Although single kinetic descriptions exist for special cases of this problem, there are no simple general prediction schemes. In this work, we have measured experimentally, with no fluorescent labelling, the displacement of an oligonucleotide from its substrate in two situations: one corresponding to oligonucleotide binding/unbinding on ssDNA and one in which the oligonucleotide is displaced by the refolding of a dsDNA fork.

Resonances at Fundamental and Harmonic Frequencies for Selective Imaging of Sine-Wave Illuminated Reversibly Photoactivatable Labels.

We introduce HIGHLIGHT as a simple and general strategy to selectively image a reversibly photoactivatable fluorescent label associated with a given kinetics. The label is submitted to sine-wave illumination of large amplitude, which generates oscillations of its concentration and fluorescence at higher harmonic frequencies. For singularizing a label, HIGHLIGHT uses specific frequencies and mean light intensities associated with resonances of the amplitudes of concentration and fluorescence oscillations at harmonic frequencies.