Measuring Direct Forces on dsRNA in Solid State Nanopores

Abstract

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.