Troy A. Lionberger

siRNA-Like Double-Stranded RNAs Are Specifically Protected Against Degradation in Human Cell Extract

RNA interference (RNAi) is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence) any gene of interest by the introduction of synthetic small-interfering (si)RNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras) to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand) double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time.

Transcription factors IIS and IIF enhance transcription efficiency by differentially modifying RNA polymerase pausing dynamics

Significance Regulation of gene expression controls fundamental cellular processes, such as growth and differentiation. Gene expression begins at transcription, in which the eukaryotic RNA polymerase (Pol II) synthesizes the precursors of mRNA during the elongation phase. The speed and fidelity of the process are regulated by many transcription elongation factors, including transcription factors IIS (TFIIS) and IIF (TFIIF), which directly interact with the enzyme. Using a single-molecule optical-tweezers assay, we have quantified the effects of these factors on the dynamics of transcription elongation by Pol II on both bare and nucleosomal DNA. We showed that TFIIF mainly prevents Pol II from pause entering, whereas TFIIS assists with pause recovery, and quantitatively described these effects on the kinetics of transcription elongation by Pol II. Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to stimulate transcription elongation. Here, we use a single-molecule transcription elongation assay to study the effects of both factors. We find that these transcription factors enhance overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observe that both factors enhance the processivity of RNA polymerase II through the nucleosomal barrier. The effects of TFIIS and TFIIF are quantitatively described using the linear Brownian ratchet kinetic model for transcription elongation and the backtracking model for transcriptional pauses, modified to account for the effects of the transcription factors. Our findings help elucidate the molecular mechanisms by which transcription factors modulate gene expression.

Cooperative kinking at distant sites in mechanically stressed DNA

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA.

Structural Ensemble and Dynamics of Toroidal-like DNA Shapes in Bacteriophage ϕ29 Exit Cavity

In the bacteriophage ϕ29, DNA is packed into a preassembled capsid from which it ejects under high pressure. A recent cryo-EM reconstruction of ϕ29 revealed a compact toroidal DNA structure (30-40 basepairs) lodged within the exit cavity formed by the connector-lower collar protein complex. Using multiscale models, we compute a detailed structural ensemble of intriguing DNA toroids of various lengths, all highly compatible with experimental observations. In particular, coarse-grained (elastic rod) and atomistic (molecular dynamics) models predict the formation of DNA toroids under significant compression, a largely unexplored state of DNA. Model predictions confirm that a biologically attainable compressive force of 25 pN sustains the toroid and yields DNA electron density maps highly consistent with the experimental reconstruction. The subsequent simulation of dynamic toroid ejection reveals large reactions on the connector that may signal genome release.

Bending the Rules of Transcriptional Repression: Tightly Looped DNA Directly Represses T7 RNA Polymerase

From supercoiled DNA to the tight loops of DNA formed by some gene repressors, DNA in cells is often highly bent. Despite evidence that transcription by RNA polymerase (RNAP) is affected in systems where DNA is deformed significantly, the mechanistic details underlying the relationship between polymerase function and mechanically stressed DNA remain unclear. Seeking to gain additional insight into the regulatory consequences of highly bent DNA, we hypothesize that tightly looping DNA is alone sufficient to repress transcription. To test this hypothesis, we have developed an assay to quantify transcription elongation by bacteriophage T7 RNAP on small, circular DNA templates approximately 100 bp in size. From these highly bent transcription templates, we observe that the elongation velocity and processivity can be repressed by at least two orders of magnitude. Further, we show that minicircle templates sustaining variable levels of twist yield only moderate differences in repression efficiency. We therefore conclude that the bending mechanics within the minicircle templates dominate the observed repression. Our results support a model in which RNAP function is highly dependent on the bending mechanics of DNA and are suggestive of a direct, regulatory role played by the template itself in regulatory systems where DNA is known to be highly bent.

Recording single motor proteins in the cytoplasm of mammalian cells.

Biomolecular motors are central to the function and regulation of all cellular transport systems. The molecular mechanisms by which motors generate force and motion along cytoskeletal filaments have been mostly studied in vitro using a variety of approaches, including several single-molecule techniques. While such studies have revealed significant insights into the chemomechanical transduction mechanisms of motors, important questions remain unanswered as to how motors work in cells. To understand how motor activity is regulated and how motors orchestrate the transport of specific cargoes to the proper subcellular domain requires analysis of motor function in vivo. Many transport processes in cells are believed to be powered by single or very few motor molecules, which makes it essential to track, in real time and with nanometer resolution, individual motors and their associated cargoes and tracks. Here we summarize, contrast, and compare recent methodological advances, many relying on advanced fluorescent labeling, genetic tagging, and imaging techniques, that lay the foundation for groundbreaking approaches and discoveries. In addition, to illustrate the impact and capabilities for these methods, we highlight novel biological findings where appropriate.

Highly sensitive ratiometric quantification of cyanide in water with gold nanoparticles via Resonance Rayleigh Scattering

A highly sensitive and selective ratiometric sensor for the quantification of cyanide (CN-) in aqueous samples has been developed using spherical gold nanoparticles (AuNPs) stabilized by polysorbate 40 (PS-40). Three different AuNP sizes (14, 40 and 80nm mean diameters) were used to evaluate the response of the sensor using both colorimetric and Resonance Rayleigh Scattering (RRS) detection schemes. The best results were obtained for the sensor using 40nm AuNPs, for which the limits of detection (LODs) were found to be 100nmolL-1 in a benchtop instrument and 500nmolL-1 by the naked eye, values well below the maximum acceptable level for drinking water (1.9µmolL-1) set by the World Health Organization (WHO). The practical use of the 40nm-AuNPs RRS sensor was demonstrated with the determination of CN- in drinking and fresh waters. Finally, the sensor was successfully implemented in a compact portable device consisting of two light-emitting diodes (LEDs) and a miniature spectrometer, turning this sensor into a very potent tool for its application as a quick routine field-deployable analytical method.

Two-Color, Laser Excitation Improves Temporal Resolution for Detecting the Dynamic, Plasmonic Coupling between Metallic Nanoparticles

The ability of two, scattering gold nanoparticles (GNPs) to plasmonically couple in a manner that is dependent on the interparticle separation has been exploited to measure nanometer-level displacements. However, despite broad applicability to monitoring biophysical dynamics, the long time scales (<5 Hz) with which plasmonic coupling are typically measured are not suitable for many dynamic molecular processes, generally occurring over several milliseconds. Here, we introduce a new technique intended to overcome this technical limitation: ratiometric analysis using monochromatic, evanescent darkfield illumination (RAMEDI). As a proof-of-principle, we monitored dynamic, plasmonic coupling arising from the binding of single biotin- and neutravidin-GNPs with a temporal resolution of 38 ms. We also show that the observable bandwidth is extendable to faster time scales by demonstrating that RAMEDI is capable of achieving a signal-to-noise ratio greater than 20 from individual GNPs observed with 200 Hz bandwidth.

Fabrication of nanoscale zero-mode waveguides using microlithography for single molecule sensing

We present a novel approach to the fabrication of zero-mode waveguides (ZMWs) using inexpensive processing techniques. Our method is capable of rapid fabrication of circular nanoapertures with diameters ranging from 70 nm to 2 μm, allowing us to perform a detailed characterization of the dependence of the fluorescence emission on the waveguide diameter. We also validated the use of the fabricated ZMWs by detecting single molecule binding events with a signal-to-noise ratio of ten.

Full molecular trajectories of RNA polymerase at single base-pair resolution

Significance Optical tweezers enable scientists to follow the dynamics of molecular motors at high resolution. The ability to discern a motor’s discrete steps reveals important insights on its operation. Some motors operate at the scale of angstroms, rendering the observation of their steps extremely challenging. In some cases, such small steps have been observed sporadically; however, the full molecular trajectories of steps and intervals between steps remain elusive due to instrumental noise. Here, we eliminate the main source of noise of most high-resolution dual-trap optical tweezers and developed both a single-molecule assay and a self-learning algorithm to uncover the full trajectories of such a motor: RNA polymerase. Using this method, a whole new set of experiments becomes possible. In recent years, highly stable optical tweezers systems have enabled the characterization of the dynamics of molecular motors at very high resolution. However, the motion of many motors with angstrom-scale dynamics cannot be consistently resolved due to poor signal-to-noise ratio. Using an acousto-optic deflector to generate a “time-shared” dual-optical trap, we decreased low-frequency noise by more than one order of magnitude compared with conventional dual-trap optical tweezers. Using this instrument, we implemented a protocol that synthesizes single base-pair trajectories, which are used to test a Large State Space Hidden Markov Model algorithm to recover their individual steps. We then used this algorithm on real transcription data obtained in the same instrument to fully uncover the molecular trajectories of Escherichia coli RNA polymerase. We applied this procedure to reveal the effect of pyrophosphate on the distribution of dwell times between consecutive polymerase steps.