Michiel van den Hout

Distinguishing Single- and Double-Stranded Nucleic Acid Molecules Using Solid-State Nanopores

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.

Controlling nanopore size, shape and stability

Solid-state nanopores are considered a promising tool for the study of biological polymers such as DNA and RNA, due largely to their flexibility in size, potential in device integration and robustness. Here, we show that the precise shape of small nanopores (approximately 5 nm diameter in 20 nm SiN membranes) can be controlled by using transmission electron microscope (TEM) beams of different sizes. However, when some of these small nanopores are immersed in an aqueous solution, their resistance is observed to decrease over time. By comparing nanopores of different shapes using (scanning) TEM both before and after immersion in aqueous solution, we demonstrate that the stability of small nanopores is related to their three-dimensional geometry, which depends on the TEM beam size employed during pore fabrication. Optimal stability is obtained using a TEM beam size of approximately the same size as the intended nanopore diameter. In addition, we show that thermal oxidation can serve as a means to independently control nanopore size following TEM fabrication. These observations provide key guidelines for the fabrication of stable solid-state nanopores on the scale of nucleic acids and small proteins.

Direct force measurements on double-stranded RNA in solid-state nanopores.

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.

Distinguishable Populations Report on the Interactions of Single DNA Molecules with Solid-State Nanopores

Solid-state nanopores have received increasing interest over recent years because of their potential for genomic screening and sequencing. In particular, small nanopores (2-5 nm in diameter) allow the detection of local structure along biological molecules, such as proteins bound to DNA or possibly the secondary structure of RNA molecules. In a typical experiment, individual molecules are translocated through a single nanopore, thereby causing a small deviation in the ionic conductance. A correct interpretation of these conductance changes is essential for our understanding of the process of translocation, and for further sophistication of this technique. Here, we present translocation measurements of double-stranded DNA through nanopores down to the diameter of the DNA itself (1.8-7 nm at the narrowest constriction). In contrast to previous findings on such small nanopores, we find that single molecules interacting with these pores can cause three distinct levels of conductance blockades. We attribute the smallest conductance blockades to molecules that briefly skim the nanopore entrance without translocating, the intermediate level of conductance blockade to regular head-to-tail translocations, and the largest conductance blockades to obstruction of the nanopore entrance by one or multiple (duplex) DNA strands. Our measurements are an important step toward understanding the conductance blockade of biomolecules in such small nanopores, which will be essential for future applications involving solid-state nanopores.

An RNA toolbox for single-molecule force spectroscopy studies

Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNA-enzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct. Such a construct is typically subject to several criteria. First, single-molecule force spectroscopy techniques often require an RNA construct that is longer than the RNA molecules used for bulk biochemical studies. Next, the incorporation of modified nucleotides into the RNA construct is required for its surface immobilization. In addition, RNA constructs for single-molecule studies are commonly assembled from different single-stranded RNA molecules, demanding good control of hybridization or ligation. Finally, precautions to prevent RNase- and divalent cation-dependent RNA digestion must be taken. The rather limited selection of molecular biology tools adapted to the manipulation of RNA molecules, as well as the sensitivity of RNA to degradation, make RNA construct preparation a challenging task. We briefly illustrate the types of single-molecule force spectroscopy experiments that can be performed on RNA, and then present an overview of the toolkit of molecular biology techniques at ones disposal for the assembly of such RNA constructs. Within this context, we evaluate the molecular biology protocols in terms of their effectiveness in producing long and stable RNA constructs.

Scanning tunneling spectroscopy on a carbon nanotube buckle

The topography and electronic structure of a semiconducting carbon nanotube with a sharp bend is studied by scanning tunneling microscopy and spectroscopy. From the increased height it is concluded that the nanotube is “buckled,” i.e. collapsed due to very strong bending. Detailed spatially resolved spectroscopy measurements at the location of the buckle reveal a well-defined localized state inside the semiconducting gap. The spatial extent of this localized state is about 2 nanometers.

Measuring Direct Forces on dsRNA in Solid State Nanopores

In recent years, far-reaching discoveries about the functionality of RNA in biology have been made. Especially double stranded RNA (dsRNA) is found to play a key role in the process of RNA interference. We employ solid state nanopores (nanometer sized holes in a thin SiN membrane) to study single RNA molecules. By applying an electrical field over the nanopore, RNA molecules can be threaded into the nanopore, causing a change in the ionic current. This change can provide insight into some of their structural properties, such as charge density, diameter, and possibly also their local structure. We have integrated our nanopore setup with optical tweezers, which allows us to also measure and apply forces to the molecule inside the nanopore.Here, we present the first application of this new technique to the study of RNA molecules, in this case long dsRNA. We show that the force experienced on these molecules is very similar to that on DNA molecules, as one would expect from the very similar structure of these molecules. In addition, we show that the measured force is independent on the distance of the optical trap to the nanopore, even at very close range (< 500 nm). Measuring forces at such close distances may be required for the application of this technique to more complicated molecules, such as single stranded RNA molecules or RNA-protein complexes. Finally, we have further extended the use of this technique to very small nanopores (down to ∼3 nm in diameter), also an important future requirement to study more complex molecules. Combined, these measurements represent important steps towards the detection of local structure along RNA molecules.