by scDNA technology
"We devised scDNA as robust, nick-free double-stranded templates for easy and reproducible single-molecule micromanipulation. They are compatible with most current setups, whether one relies on magnetic, optical, acoustic, or centrifugal forces. scDNA are available in two different lengths so as to better suit users’ requirements in terms of handling and resolution. Furthermore, their ends have been multiply functionalized with biotin or digoxigenin, enabling long-term engraftment on glass slides or beads coated with streptavidin or anti-digoxigenin. With these molecules in hand, exploring the mechanics of DNA and the interactions of nucleic acids with proteins is straightforward."
EXAMPLES OF RESULTS
Dynatwist are supercoilable double-stranded DNA molecules with exceptional stability and high purity (> 95%).
(a) A 17 kbp (biotin, digoxigenin) scDNA molecule is micromanipulated thanks to a micron-size magnetic bead. The set of magnets located above the sample is rotated by a given number of turns n to precisely control the applied torsional stress whilst it is also translated vertically to modulate the applied longitudinal stress. Writhed plectonemic structures only appear at low force, resulting in a bead very close to the glass surface. (a’) Time trace for DNA extension obtained when n = + 64 and when the magnetic force is alternated between Flow = 0.3 pN and Fhigh = 5 pN. (b) In absence of ATP topoisomerase II from D. melanogaster binds adjacent DNA segments and forms a stable ternary complexe. Therefore, removing supercoil by traction is only possible up to a certain extent. (b’) Time trace for DNA extension obtained in the same condition than above, topoisomerases now forming clamps that limit the upward motion of the bead when the force is increased. Eventually, some of the ternary complexes dissociate during the pulling phase and jumps in extension can be observed.
(a) A 5 kbp (biotin, digoxigenin) scDNA molecule is micromanipulated thanks to a 1 μm diameter magnetic bead. The set of magnets located above the sample is rotated by n = - 16 turns and a constant pulling force is applied, Fmag = 0.45 pN. With such a negative supercoiling writhed plectonemic structures appear, that can transiently disappear went an inverted repeat converts into a cruciform motif. The latter topological transformation results in a large upward motion of the bead. (a’) Corresponding time trace for DNA extension. (b) Histogram of the transition amplitudes. The arrow head indicates the position of the mean <Δℓ> = 241 ± 3 nm. (c) Histograms of the dwell times and single-exponential fits to the data. The characteristic time for cruciform folding is τfolding = 20.2 ± 1.2 s and the one for cruciform unfolding τunfolding = 13.5 ± 0.9 s.
Data have been acquired on a homemade magnetic tweezers setup by T. Ramreddy and T. Strick (Institut Jacques Monod, Paris, France).
An extreme quality: Less than 5 % of the provided double-stranded DNA molecules present a nick in one of their phosphate backbones (cf. batch characterization report)
Two long and multiply labeled extremities: Each extremity includes multiple digoxigenin or biotin functional groups over a controlled length of 1 kbp, which ensures robust and long-lasting engraftment to beads and surfaces
A central fragment of precisely defined length: The unlabeled part of the construct is either 5 and 17 kbp long, so as to fulfill needs in high-sensitivity detection of molecular interactions or high-precision force calibration