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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98608/1/0957-4484_23_45_455301.pd

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 DN

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

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

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