Revisiting the central dogma one molecule at a time.

The faithful relay and timely expression of genetic information depend on specialized molecular machines, many of which function as nucleic acid translocases. The emergence over the last decade of single-molecule fluorescence detection and manipulation techniques with nm and Å resolution and their application to the study of nucleic acid translocases are painting an increasingly sharp picture of the inner workings of these machines, the dynamics and coordination of their moving parts, their thermodynamic efficiency, and the nature of their transient intermediates. Here we present an overview of the main results arrived at by the application of single-molecule methods to the study of the main machines of the central dogma.

Aberration-corrected cryoimmersion light microscopy.

Cryogenic fluorescent light microscopy of flash-frozen cells stands out by artifact-free fixation and very little photobleaching of the fluorophores used. To attain the highest level of resolution, aberration-free immersion objectives with accurately matched immersion media are required, but both do not exist for imaging below the glass-transition temperature of water. Here, we resolve this challenge by combining a cryoimmersion medium, HFE-7200, which matches the refractive index of room-temperature water, with a technological concept in which the body of the objective and the front lens are not in thermal equilibrium. We implemented this concept by replacing the metallic front-lens mount of a standard bioimaging water immersion objective with an insulating ceramic mount heated around its perimeter. In this way, the objective metal housing can be maintained at room temperature, while creating a thermally shielded cold microenvironment around the sample and front lens. To demonstrate the range of potential applications, we show that our method can provide superior contrast in Escherichia coli and yeast cells expressing fluorescent proteins and resolve submicrometer structures in multicolor immunolabeled human bone osteosarcoma epithelial (U2OS) cells at [Formula: see text]C.

Trigger loop folding determines transcription rate of Escherichia coli's RNA polymerase.

Two components of the RNA polymerase (RNAP) catalytic center, the bridge helix and the trigger loop (TL), have been linked with changes in elongation rate and pausing. Here, single molecule experiments with the WT and two TL-tip mutants of the Escherichia coli enzyme reveal that tip mutations modulate RNAP's pause-free velocity, identifying TL conformational changes as one of two rate-determining steps in elongation. Consistent with this observation, we find a direct correlation between helix propensity of the modified amino acid and pause-free velocity. Moreover, nucleotide analogs affect transcription rate, suggesting that their binding energy also influences TL folding. A kinetic model in which elongation occurs in two steps, TL folding on nucleoside triphosphate (NTP) binding followed by NTP incorporation/pyrophosphate release, quantitatively accounts for these results. The TL plays no role in pause recovery remaining unfolded during a pause. This model suggests a finely tuned mechanism that balances transcription speed and fidelity.

Microfluidic cryofixation for correlative microscopy.

Cryofixation yields outstanding ultrastructural preservation of cells for electron microscopy, but current methods disrupt live cell imaging. Here we demonstrate a microfluidic approach that enables cryofixation to be performed directly in the light microscope with millisecond time resolution and at atmospheric pressure. This will provide a link between imaging/stimulation of live cells and post-fixation optical, electron, or X-ray microscopy.

A fluorescence based method for the quantification of surface functional groups in closed micro- and nanofluidic channels.

Surface analysis is critical for the validation of microfluidic surface modifications for biology, chemistry, and physics applications. However, until now quantitative analytical methods have mostly been focused on open surfaces. Here, we present a new fluorescence imaging method to directly measure the surface coverage of functional groups inside assembled microchannels over a wide dynamic range. A key advance of our work is the elimination of self-quenching to obtain a linear signal even with a high density of functional groups. This method is applied to image the density and monitor the stability of vapor deposited silane layers in bonded silicon/glass micro- and nanochannels.

Thermal probing of E. coli RNA polymerase off-pathway mechanisms.

RNA polymerase (RNAP) is an essential enzyme for cellular gene expression. In an effort to further understand the enzyme's importance in the cell's response to temperature, we have probed the kinetic mechanism of Escherichia coli RNAP by studying the force-velocity behavior of individual RNAP complexes at temperatures between 7 and 45 degrees C using optical tweezers. Within this temperature range and at saturating nucleotide concentrations, the pause-free transcription velocity of RNAP was independent of force and increased monotonically with temperature with an elongation activation energy of 9.7+/-0.7 kcal/mol. Interestingly, the pause density at cold temperatures (7 to 21 degrees C) was five times higher than that measured above room temperature. A simple kinetic model revealed a value of 1.29+/-0.05 kcal/mol for the activation energy of pause entry, suggesting that pause entry is indeed a thermally accessible process. The dwell time distribution of all observable pauses was independent of temperature, directly confirming a prediction of the model recently proposed for Pol II in which pauses are diffusive backtracks along the DNA. Additionally, we find that the force at which the polymerase arrests (the arrest force) presents a maximum at 21 degrees C, an unexpected result as this is not the optimum temperature for bacterial growth. This observation suggests that arrest could play a regulatory role in vivo, possibly through interactions with specific elongation factors.

Nanophotonic crescent moon structures with sharp edge for ultrasensitive biomolecular detection by local electromagnetic field enhancement effect.

We present novel gold nanophotonic crescent moon structures with a sub-10 nm sharp edge, which can enhance local electromagnetic field at the edge area. The formation of unconventional nanophotonic crescent moon structure is accomplished by using a sacrificial nanosphere template and conventional thin film deposition method, which allows an effective batch nanofabrication and precise controls of nanostructure shapes. Unique multiple scattering peaks are observed in a single gold nanocrescent moon with dark-field white light illumination. A 785 nm near-infrared (NIR) diode laser was used as the excitation source to induce the amplified scattering field on the sharp edge of the single gold nanocrescent moon. The Raman scattering spectrum of Rhodamine 6G molecules adsorbed on the single gold nanocrescent moon are characterized, and the Raman enhancement factor of single gold nanocrescent moon is estimated larger than 10(10), which suggests the potential applications of gold nanocrescent moons in ultrasensitive biomolecular detection and cellular imaging using surface enhanced Raman spectroscopy.