Nynke Dekker

Abstract:

The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in the bacterial cell cycle and virulence. RelA integrates several environmental cues and synthesizes the alarmone ppGpp, which globally reprograms transcription, translation, and replication. We have developed and implemented novel single-molecule tracking methodology to characterize the intracellular catalytic cycle of RelA. Our single-molecule experiments show that RelA is on the ribosome under nonstarved conditions and that the individual enzyme molecule stays off the ribosome for an extended period of time after activation. This suggests that the catalytically active part of the RelA cycle is performed off, rather than on, the ribosome, and that rebinding to the ribosome is not necessary to trigger each ppGpp synthesis event. Furthermore, we find fast activation of RelA in response to heat stress followed by RelA rapidly being reset to its inactive state, which makes the system sensitive to new environmental cues and hints at an underlying excitable response mechanism.

Abstract:

Solid-state nanopores are widely acknowledged as tools with which to study local structure in biological molecules. Individual molecules are forced through a nanopore, causing a characteristic change in an ionic current that depends on the molecules' local diameter and charge distribution. Here, the translocation measurements of long (~5-30 kilobases) single-stranded poly(U) and poly(A) molecules through nanopores ranging from 1.5 to 8 nm in diameter are presented. Individual molecules are found to be able to cause multiple levels of conductance blockade upon traversing the pore. By analyzing these conductance blockades and their relative incidence as a function of nanopore diameter, it is concluded that the smallest conductance blockades likely correspond to molecules that translocate through the pore in predominantly head-to-tail fashion. The larger conductance blockades are likely caused by molecules that arrive at the nanopore entrance with many strands simultaneously. These measurements constitute the first demonstration that single-stranded RNA can be captured in solid-state nanopores that are smaller than the diameter of double-stranded RNA. These results further the understanding of the conductance blockades caused by nucleic acids in solid-state nanopores, relevant for future applications, such as the direct determination of RNA secondary structure.

Abstract:

Numerous biological and biotechnological applications rely on the use of micrometer- and nanometer-scale particles, benefiting tremendously from quantitative control of their physical and chemical properties. Here, we describe the use of electron beam lithography for the design, fabrication, and functionalization of micrometer-scale birefringent quartz cylinders for use in sensing and detection. We demonstrate excellent control of the cylinders' geometry, fabricating cylinders with heights of 0.5-2 μm and diameters of 200-500 nm with high precision while maintaining control of their side-wall angle. The flexible fabrication allows cylinders to be selectively shaped into conical structures or to include centered protrusions for the selective attachment of biomolecules. The latter is facilitated by straightforward functionalization targeted either to a cylinder's face or to the centered protrusion alone. The fabricated quartz cylinders are characterized in an optical torque wrench, permitting correlation of their geometrical properties to measured torques. Lastly, we tether individual DNA molecules to the functionalized cylinders and demonstrate the translational and rotational control required for single-molecule studies.

Abstract:

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.