Gary M. Skinner

RNA plays myriad roles in the transmission and regulation of genetic information that are fundamentally constrained by its mechanical properties, including the elasticity and conformational transitions of the double-stranded (dsRNA) form. Although double-stranded DNA (dsDNA) mechanics have been dissected with exquisite precision, much less is known about dsRNA. Here we present a comprehensive characterization of dsRNA under external forces and torques using magnetic tweezers. We find that dsRNA has a force-torque phase diagram similar to that of dsDNA, including plectoneme formation, melting of the double helix induced by torque, a highly overwound state termed "P-RNA," and a highly underwound, left-handed state denoted "L-RNA." Beyond these similarities, our experiments reveal two unexpected behaviors of dsRNA: Unlike dsDNA, dsRNA shortens upon overwinding, and its characteristic transition rate at the plectonemic buckling transition is two orders of magnitude slower than for dsDNA. Our results challenge current models of nucleic acid mechanics, provide a baseline for modeling RNAs in biological contexts, and pave the way for new classes of magnetic tweezers experiments to dissect the role of twist and torque for RNA-protein interactions at the single-molecule level.

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.

We have found strong supporting evidence for the helical structures of single-stranded nucleic acids by stretching individual molecules of polyadenylic acid [poly(A)] and polycytidylic acid [poly(C)]. Analyzing the force versus extension data using a two-state elastic model in which random-coil domains alternate with rigid helical domains allows one to extract the thermodynamic and structural properties. In addition, it also yields moderate to low cooperativity of the helix-coil transition for poly(A) and poly(C), respectively.

We have investigated the elastic properties of poly(U), homopolymeric single-stranded RNA molecules that lack any base pairing and stacking interactions and conform to a random-coil structure. Using single-molecule stretching experiments we show that the elastic properties are described by a wormlike chain model for polymer elasticity rather than by a freely jointed chain model as is commonly used for single-stranded DNA. At low [Na+], introduction of a scale-dependent persistence length is required to account for electrostatic contributions.

A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k(off)= 2.9 s(-1)) of T7 RNAP and promoter DNA, the transition to the elongation mode (k(for) = 0.36 s(-1)), and the processive synthesis (k(pol) = 43 nt s(-1)) and release of a gene-length RNA transcript ( approximately 1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k(off) > k(for)), identifying a rate-limiting step between promoter DNA binding and promoter escape.