Nynke Dekker

Abstract:

Solid-state nanopores can be employed to detect and study local structure along single molecules by voltage driven translocation through the nanopore. Their sensitivity and versatility can be augmented by combining them with a direct force probe, for example, optical tweezers. Such a tool could potentially be used to directly probe RNA secondary structure through the sequential unfolding of duplex regions. Here, we demonstrate the first application of such a system to the study of RNA by directly measuring the net force on individual double-stranded RNA (dsRNA) molecules. We have probed the force on dsRNA over a large range of nanopore sizes from 35 nm down to 3.5 nm and find that it decreases as the pore size is increased, in accordance with numerical calculations. Furthermore, we find that the force is independent of the distance between the optical trap and the nanopore surface, permitting force measurement on quite short molecules. By comparison with dsDNA molecules trapped in the same nanopores, we find that the force on dsRNA is on the order of, but slightly lower than, that on dsDNA. With these measurements, we expand the possibilities of the nanopore-optical tweezers to the study of RNA molecules with potential applications to the detection of RNA-bound proteins, the determination of RNA secondary structure, and the processing of RNA by molecular motors.

Abstract:

We report translocation of double-stranded DNA (dsDNA) molecules that are coated with RecA protein through solid-state nanopores. Translocation measurements show current-blockade events with a wide variety in time duration (10-4-10-1 s) and conductance blockade values (3-14 nS). Large blockades (11.4+/-0.7 nS) are identified as being caused by translocations of RecA-dsDNA filaments. We confirm these results through a variety of methods, including changing molecular length and using an optical tweezer system to deliver bead-functionalized molecules to the nanopore. We further distinguish two different regimes of translocation: a low-voltage regime (<150 mV) in which the event rate increases exponentially with voltage, and a high-voltage regime in which it remains constant. Our results open possibilities for a variety of future experiments with (partly) protein-coated DNA molecules, which is interesting for both fundamental science and genomic screening applications.

Abstract:

Solid-state nanopores offer a promising method for rapidly probing the structural properties of biopolymers such as DNA and RNA. We have for the first time translocated RNA molecules through solid-state nanopores, comparing the signatures of translocating double-stranded RNA molecules and of single-stranded homopolymers poly(A), poly(U), poly(C). On the basis of their differential blockade currents, we can rapidly discriminate between both single- and double-stranded nucleic-acid molecules, as well as separate purine-based homopolymers from pyrimidine-based homopolymers. Molecule identification is facilitated through the application of high voltages ( approximately 600 mV), which contribute to the entropic stretching of these highly flexible molecules. This striking sensitivity to relatively small differences in the underlying polymer structure greatly improves the prospects for using nanopore-based devices for DNA or RNA mapping.

Abstract:

Magnetic tweezers are a powerful tool to manipulate single DNA or RNA molecules and to study nucleic acid-protein interactions in real time. Here, we have modeled the magnetic fields of permanent magnets in magnetic tweezers and computed the forces exerted on superparamagnetic beads from first principles. For simple, symmetric geometries the magnetic fields can be calculated semianalytically using the Biot-Savart law. For complicated geometries and in the presence of an iron yoke, we employ a finite-element three-dimensional PDE solver to numerically solve the magnetostatic problem. The theoretical predictions are in quantitative agreement with direct Hall-probe measurements of the magnetic field and with measurements of the force exerted on DNA-tethered beads. Using these predictive theories, we systematically explore the effects of magnet alignment, magnet spacing, magnet size, and of adding an iron yoke to the magnets on the forces that can be exerted on tethered particles. We find that the optimal configuration for maximal stretching forces is a vertically aligned pair of magnets, with a minimal gap between the magnets and minimal flow cell thickness. Following these principles, we present a configuration that allows one to apply > or = 40 pN stretching forces on approximately 1-microm tethered beads.