Molecular force balance measurements reveal that double-stranded DNA unbinds under force in rate-dependent pathways.

Strand separation of double-stranded DNA is a crucial step for essential cellular processes such as recombination and transcription. By means of a molecular force balance, we have analyzed the impact of different pulling directions and different force-loading rates on the unbinding process of short double-stranded DNA. At loading rates above 9 x 10(5) pN/s, we found a marked difference in rupture probability for pulling the duplex in 3'-3' direction compared to a 5'-5' direction, indicating different unbinding pathways. We propose a mechanism by which unbinding at low loading rates is dominated by nondirectional thermal fluctuations, whereas mechanical properties of the DNA become more important at high loading rates and reveal the asymmetry of the phosphoribose backbone. Our model explains the difference of 3'-3' and 5'-5' unbinding as a kinetic process, where the loading rate exceeds the relaxation time of DNA melting bubbles.

Print your atomic force microscope.

Progress in scanning probe microscopy profited from a flourishing multitude of new instrument designs, which lead to novel imaging modes and as a consequence to innovative microscopes. Often these designs were hampered by the restrictions, which conventional milling techniques impose. Modern rapid prototyping techniques, where layer by layer is added to the growing piece either by light driven polymerization or by three-dimensional printing techniques, overcome this constraint, allowing highly concave or even embedded and entangled structures. We have employed such a technique to manufacture an atomic force microscopy (AFM) head, and we compared its performance with a copy milled from aluminum. We tested both AFM heads for single molecule force spectroscopy applications and found little to no difference in the signal-to-noise ratio as well as in the thermal drift. The lower E modulus seems to be compensated by higher damping making this material well suited for low noise and low drift applications. Printing an AFM thus offers unparalleled freedom in the design and the rapid production of application-tailored custom instruments.

Phase contrast and DIC illumination for AFM hybrids.

High-resolution optical microscopy is an essential pre-requisite for life science force microscopy, particularly for applications in cell biology and medicine. Identification and validation of cells is typically established with techniques like phase contrast microscopy or differential interference contrast microscopy. The option to select or monitor individual cells online with such light microscopy techniques while performing atomic force microscopy (AFM) measurements is therefore extremely beneficial. Here, we report two conceptually different strategies to implement these light microscopy techniques in a fully functional AFM head at the ultimate resolution of the Abbe diffraction limit.

Dynamic restacking of Escherichia coli P-pili.

P-pili of uropathogenic Escherichia coli mediate the attachment to epithelial cells in the human urinary tract and kidney and therefore play an important role in infection. A better understanding of this mechanism could help to prevent bacteria from spreading but also provides interesting insights into molecular mechanics for future nanotech applications. The helical rod design of P-pili provides an efficient design to withstand hydrodynamic shear forces. The adhesive PapG unit at the distal end of the P-pilus forms a specific bond with the glycolipid Galabiose. This bond has a potential width Deltax = 0.7 +/- 0.15 nm and a dissociation rate K (Off) = 8.0.10(-4) +/- 5.0.10(-4) s(-1). It withstands a force of approximately 49 pN under physiological conditions. Additionally, we analyzed the behavior of unstacking and restacking of the P-pilus with dynamic force spectroscopy at velocities between 200 and 7,000 nm/s. Up to a critical extension of 66% of the totally stretched P-pilus, un/re-stacking was found to be fully reversible at velocities up to 200 nm/s. If the P-pilus is stretched beyond this critical extension a characteristic hysteresis appears upon restacking. This hysteresis originates from a nucleation process comparable to a first-order phase transition in an undercooled liquid. Analysis of the measurement data suggests that 20 PapA monomers are involved in the formation of a nucleation kernel.

B-S transition in short oligonucleotides.

Stretching experiments with long double-stranded DNA molecules in physiological ambient revealed a force-induced transition at a force of 65 pN. During this transition between B-DNA and highly overstretched S-DNA the DNA lengthens by a factor of 1.7 of its B-form contour length. Here, we report the occurrence of this so-called B-S transition in short duplexes consisting of 30 basepairs. We employed atomic-force-microscope-based single molecule force spectroscopy to explore the unbinding mechanism of two short duplexes containing 30 or 20 basepairs by pulling at the opposite 5' termini. For a 30-basepair-long DNA duplex the B-S transition is expected to cause a length increase of 6.3 nm and should therefore be detectable. Indeed 30% of the measured force-extension curves exhibit a region of constant force (plateau) at 65 pN, which corresponds to the B-S transition. The observed plateaus show a length between 3 and 7 nm. This plateau length distribution indicates that the dissociation of a 30-basepair duplex mainly occurs during the B-S transition. In contrast, the measured force-extension curves for a 20-basepair DNA duplex exhibited rupture forces below 65 pN and did not show any evidence of a B-S transition.