Chi-Fu Yen

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis

A simple genetic tag-based labeling method that permits specific attachment of a fluorescence probe near the C terminus of virtually any subunit of a protein complex is implemented. Its immediate application to yeast RNA polymerase II (pol II) enables us to test various hypotheses of RNA exit channel by using fluorescence resonance energy transfer (FRET) analysis. The donor dye is labeled on a site near subunit Rpb3 or Rpb4, and the acceptor dye is attached to the 5′ end of RNA transcript in the pol II elongation complex. Both in-gel and single-molecule FRET analysis show that the growing RNA is leading toward Rpb4, not Rpb3, supporting the notion that RNA exits through the proposed channel 1. Distance constraints derived from our FRET results, in conjunction with triangulation, reveal the exit track of RNA transcript on core pol II by identifying amino acids in the vicinity of the 5′ end of RNA and show that the extending RNA forms contacts with the Rpb7 subunit. The significance of RNA exit route in promoter escape and that in cotranscriptional mRNA processing is discussed.

Copper-induced structural conversion templates prion protein oligomerization and neurotoxicity

Copper induces prion protein misfolding, aggregation, and neurotoxicity. Prion protein (PrP) misfolding and oligomerization are key pathogenic events in prion disease. Copper exposure has been linked to prion pathogenesis; however, its mechanistic basis is unknown. We resolve, with single-molecule precision, the molecular mechanism of Cu2+-induced misfolding of PrP under physiological conditions. We also demonstrate that misfolded PrPs serve as seeds for templated formation of aggregates, which mediate inflammation and degeneration of neuronal tissue. Using a single-molecule fluorescence assay, we demonstrate that Cu2+ induces PrP monomers to misfold before oligomer assembly; the disordered amino-terminal region mediates this structural change. Single-molecule force spectroscopy measurements show that the misfolded monomers have a 900-fold higher binding affinity compared to the native isoform, which promotes their oligomerization. Real-time quaking-induced conversion demonstrates that misfolded PrPs serve as seeds that template amyloid formation. Finally, organotypic slice cultures show that misfolded PrPs mediate inflammation and degeneration of neuronal tissue. Our study establishes a direct link, at the molecular level, between copper exposure and PrP neurotoxicity.

Fluorescence axial localization with nanometer accuracy and precision

We describe a new technique, standing wave axial nanometry (SWAN), to image the axial location of a single nanoscale fluorescent object with sub-nanometer accuracy and 3.7 nm precision. A standing wave, generated by positioning an atomic force microscope tip over a focused laser beam, is used to excite fluorescence; axial position is determined from the phase of the emission intensity. We use SWAN to measure the orientation of single DNA molecules of different lengths, grafted on surfaces with different functionalities.

Improvement in Resolution of Laser Capture Microdissection Using Near-Field Probe to Capture Nanoparticles

Purpose: A modified laser capture micro-dissection (LCM) system is developed to improve resolution to 400 nm, using a laser light (808 nm) transmitted by a near-field tip probe. Materials and methods: Using a 150-nm aperture to heat an ethylene vinyl acetate (EVA) film, melted spots on the average of 400 nm in diameter are generated on the underlying target composed of a 20-nm gold-particle monolayer. The near-field tip probe composed of fiber is set on a 2-D nanometer piezoactuator (PZT) for precise capturing of the monolayer of gold particles. The monolayer of gold particles under the target is bound to the EVA film using a laser, while the remaining EVA film stays on the monolayer. Results: The diameter of the melted spots as small as 400 nm are produced and details are provided that demonstrate the feasibility of the nano-operation of this new LCM system. Conclusion: The new LCM system successfully captures nanoparticles and improves resolution of micro-dissection to 400 nm. With this LCM system, the isolation of a single organelle or bacterium is possible.

Improving estimation of kinetic parameters in dynamic force spectroscopy using cluster analysis

Dynamic Force Spectroscopy (DFS) is a widely used technique to characterize the dissociation kinetics and interaction energy landscape of receptor-ligand complexes with single-molecule resolution. In an Atomic Force Microscope (AFM)-based DFS experiment, receptor-ligand complexes, sandwiched between an AFM tip and substrate, are ruptured at different stress rates by varying the speed at which the AFM-tip and substrate are pulled away from each other. The rupture events are grouped according to their pulling speeds, and the mean force and loading rate of each group are calculated. These data are subsequently fit to established models, and energy landscape parameters such as the intrinsic off-rate (koff) and the width of the potential energy barrier (xβ) are extracted. However, due to large uncertainties in determining mean forces and loading rates of the groups, errors in the estimated koff and xβ can be substantial. Here, we demonstrate that the accuracy of fitted parameters in a DFS experiment can be dramatically improved by sorting rupture events into groups using cluster analysis instead of sorting them according to their pulling speeds. We test different clustering algorithms including Gaussian mixture, logistic regression, and K-means clustering, under conditions that closely mimic DFS experiments. Using Monte Carlo simulations, we benchmark the performance of these clustering algorithms over a wide range of koff and xβ, under different levels of thermal noise, and as a function of both the number of unbinding events and the number of pulling speeds. Our results demonstrate that cluster analysis, particularly K-means clustering, is very effective in improving the accuracy of parameter estimation, particularly when the number of unbinding events are limited and not well separated into distinct groups. Cluster analysis is easy to implement, and our performance benchmarks serve as a guide in choosing an appropriate method for DFS data analysis.

Minimizing open-loop piezoactuator nonlinearity artifacts in atomic force microscope measurements

Atomic force microscopes (AFMs) are widely used to study molecular interactions with piconewton force sensitivity. In an AFM, interaction forces are measured by reflecting a laser beam off a cantilever onto a position sensitive detector and monitoring cantilever deflection. Precise measurements of interaction forces rely on accurately determining the optical lever sensitivity, i.e., the relationship between cantilever deflection and changes in detector voltage. The optical lever sensitivity is measured by pressing the cantilever against a hard substrate using a piezoactuator and recording the resulting change in detector voltage. However, nonlinearities in the motion of commonly used open-loop piezo actuators introduce significant errors in measured optical lever sensitivities. Here, the authors systematically characterize the effect of piezo actuator hysteresis and creep on errors in optical lever sensitivity and identify measurement conditions that minimize these errors.

The positional capture experiments of the LCM using near field fiber probe

Laser Capture Microdissection (LCM) is a technique that permits rapid and reliable procurement of pure population of cells from tissue sections. We created the LCM system with a near field fiber probe to transmit the laser light (808 nm) to heat the thermoplastic polymer film which was placed above the tissue section. Laser spots in nano-dimension cause the film to be melted and fused with the underlying target of choice. In the study, we set the fiber probe on a two-dimensional nanometer PZT to do precisely capture. The aperture of fiber probe we used is 100 nm through which light can be focused. We used 20 nm spread gold particles to be captured in these experiments instead of tissue sections. By moving the probe in X direction to melt spots in different distances, we can know more molecular properties of the thermoplastic polymer film especially in its resolving power. When the film is removed, the chosen particles remain bound to the film, while the rest of the tissue or particles are left behind. According to the results of this study, we can know how close the melted spots can reach to, and it is also helpful to the operation of this new LCM system.