Helen Hwang

Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity

Single-molecule FRET has been widely used for monitoring protein–nucleic acids interactions. Direct visualization of the interactions, however, often requires a site-specific labeling of the protein, which can be circuitous and inefficient. In addition, FRET is insensitive to distance changes in the 0–3-nm range. Here, we report a systematic calibration of a single molecule fluorescence assay termed protein induced fluorescence enhancement. This method circumvents protein labeling and displays a marked distance dependence below the 4-nm distance range. The enhancement of fluorescence is based on the photophysical phenomenon whereby the intensity of a fluorophore increases upon proximal binding of a protein. Our data reveals that the method can resolve as small as a single base pair distance at the extreme vicinity of the fluorophore, where the enhancement is maximized. We demonstrate the general applicability and distance sensitivity using (a) a finely spaced DNA ladder carrying a restriction site for BamHI, (b) RNA translocation by DExH enzyme RIG‐I, and (c) filament dynamics of RecA on single-stranded DNA. The high spatio-temporal resolution data and sensitivity to short distances combined with the ability to bypass protein labeling makes this assay an effective alternative or a complement to FRET.

Single-molecule imaging reveals a common mechanism shared by G-quadruplex–resolving helicases

Significance G-quadruplex (GQ) is a four-stranded DNA structure that forms in the human genome and influences gene expression and DNA replication. Stably formed GQ structures can act as a physical blockade to disrupt genomic processes. To counteract such an effect, cells possess special helicases dedicated to unfolding GQ structures. In our study, we examined three such GQ-resolving helicases, using single-molecule imaging, that show distinct binding preference to different conformations (folding directions) of GQ DNA. Nevertheless, all three GQ resolvases use the same mechanism, which involves repetitive cycles of GQ unfolding in successive runs. Such activity leads to efficient GQ hybridization with a complementary DNA and dislodging chemical GQ ligand. Our findings have implications for designing new GQ ligands for therapeutic purposes. G-quadruplex (GQ) is a four stranded DNA secondary structure that arises from a guanine rich sequence. Stable formation of GQ in genomic DNA can be counteracted by the resolving activity of specialized helicases including RNA helicase AU (associated with AU rich elements) (RHAU) (G4 resolvase 1), Bloom helicase (BLM), and Werner helicase (WRN). However, their substrate specificity and the mechanism involved in GQ unfolding remain uncertain. Here, we report that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession. Such unfolding activity of RHAU leads to efficient annealing exclusively within the same DNA molecule. The same resolving activity is sufficient to dislodge a stably bound GQ ligand, including BRACO-19, NMM, and Phen-DC3. Our study demonstrates a plausible biological scheme where different helicases are delegated to resolve specific GQ structures by using a common repetitive unfolding mechanism that provides a robust resolving power.

Single-molecule real-time detection of telomerase extension activity

The ends of eukaryotic chromosomes are capped by telomeres which consist of tandem G-rich DNA repeats stabilized by the shelterin protein complex. Telomeres shorten progressively in most normal cells due to the end replication problem. In more than 85% of cancers however, the telomere length is maintained by telomerase; a reverse transcriptase that adds telomeric TTAGGG repeats using its integral RNA template. The strong association between telomerase activity and malignancy in many cancers suggests that telomerase activity could serve as a diagnostic marker. We demonstrate single-molecule, real-time telomerase extension activity observed digitally as the telomeric repeats are added to a substrate. The human telomerase complex pulled down from mammalian cells displays extension activity dependent on dNTP concentration. In complex with the processivity factor, POT1-TPP1, telomerase adds repeats at an accelerated rate and yields longer products. Our assay provides a unique detection platform that enables the study of telomerase kinetics with single molecule resolution.

REGIMES OF PARTICLE TRAPPING IN INDUCTIVELY COUPLED PLASMA PROCESSING REACTORS

Contamination of wafers by particles in plasma processing reactors is a continuing problem affecting yields of microelectronic devices. In this letter, we report on a computational study of particle contamination of wafers in a high plasma density inductively coupled plasma (ICP) reactor. When operating with an unbiased substrate, particles readily contaminate the wafer due to high ion fluxes which produce large ion‐drag forces. Biasing the substrate with a radio frequency (rf) voltage counteracts the ion‐drag forces by increasing the opposing electrostatic forces in the sheath, thereby shielding the wafer from incoming particles. We have found three regimes of particle contamination for different ICP powers and rf biases. At high rf biases and low ICP powers, particles trap at the edge of the sheath. At low rf bias and high ICP power, ion drag forces dominate, particles do not trap, and wafer contamination is problematic. At intermediate powers and biases, particles quasitrap, leading to moderate particl...

A model for transport and agglomeration of particles in reactive ion etching plasma reactors

Dust particle contamination of wafers in reactive ion etching (RIE) plasma tools is a continuing concern in the microelectronics industry. It is common to find that particles collected on surfaces or downstream of the etch chamber are agglomerates of smaller monodisperse spherical particles. These observations, and the fact that the forces which govern the transport and trapping of particles are partly determined by their size, place importance on understanding particle growth and agglomeration mechanisms. Since individual particles in plasma etching tools are negatively charged, their agglomeration is problematic since the particles must obtain sufficient kinetic energy to overcome their mutual electrostatic repulsion. In this article, we discuss results from a model for particle agglomeration in RIE plasma tools with which we address the transport of particles and interparticle collisions resulting in agglomeration. These results indicate that the rate and extent of particle agglomeration depend on the particle density, plasma power deposition, and, to a lesser degree, gas flow. The dependence of agglomeration on rf power results from the fact that the kinetic energy of a dust particle is largely determined by its acceleration by ion drag forces. Significant agglomeration may occur in particle traps where the particle density is large.

Consequences of three-dimensional physical and electromagnetic structures on dust particle trapping in high plasma density material processing discharges

Plasma processing discharges are typically designed with the goal of having uniform reactant fluxes to the substrate and a minimum of dust particle contamination of the wafer. It is not uncommon, however, that reactors have three-dimensional (3D) structures such as antennas (or coils), gas injection nozzles, sub- or super-wafer topography and single-sided pump ports. These structures can contribute to azimuthal asymmetries in reactant fluxes. These structures may also produce dust particle traps. In this paper, a 3D plasma equipment model is applied to investigate the impact of these structures on reactant fluxes and their influence on dust particle trapping in inductively coupled radio frequency discharges under conditions where trapping is not typically obtained. We find that 3D structures, such as injection nozzles, perturb the plasma potential and ion fluxes to distances well beyond their geometrical boundaries. These perturbations are sufficient to create dust particle traps. Electromagnetic asymmetr...

Simulation of the formation of two-dimensional Coulomb liquids and solids in dusty plasmas

Dust particle transport in low-temperature plasmas has recently received considerable attention due to the desire to minimize contamination of wafers during plasma processing of microelectronics devices. Laser light scattering observations of dust particles near wafers in reactive-ion-etching (RIE) radio frequency (rf) discharges have revealed clouds which display collective behavior. These observations have motivated experimental studies of the Coulomb liquid and solid properties of these systems. In this paper, we present results from a two-dimensional model for dust particle transport in RIE rf discharges in which we include particle-particle Coulomb interactions. We predict the formation of Coulomb liquids and solids. These predictions are based both on values of Γ>2 (liquid) and Γ>170 (solid), where Γ is the ratio of electrostatic potential energy to thermal energy, and on crystal-like structure in the pair correlation function. We find that Coulomb liquids and solids composed of trapped dust particl...

Evidence for inelastic processes for N3+ and N4+ from ion energy distributions in He/N2 radio frequency glow discharges

The ion energy distributions (IEDs) striking surfaces in rf glow discharges are important in the context of plasma etching during the fabrication of microelectronics devices. In discharges sustained in molecular gases or multicomponent gas mixtures, the shape of the IED and the relative magnitudes of the ion fluxes are sensitive to ion–molecule collisions which occur in the presheath and sheath. Ions which collisionlessly traverse the sheaths or suffer only elastic collisions arrive at the substrate with a measurably different IED than do ions which undergo inelastic collisions. In this article we present measurements and results from parametric calculations of IEDs incident on the grounded electrode of a rf glow discharge sustained in a He/N2 gas mixture while using a Gaseous Electronics Conference Reference Cell (33.3 Pa, 13.56 MHz). We found that the shape of the IEDs for N+3 and N+4 provide evidence for inelastic ion–molecule reactions which have threshold energies of <10 eV.

Fluorocarbon impurities in KrF lasers

Fluorocarbon impurities are known to have deleterious effects on the operation of excimer lasers; however, the sensitivity limits are poorly known. Absorption at 248.9 nm in an e‐beam‐pumped KrF laser has been attributed to CF2, produced by plasma fragmentation of precursor molecules such as CF4. In this paper, the effects of CF4 impurities on the gain of an electron‐beam‐excited KrF laser are investigated theoretically. It is found that the density of KrF(B) significantly decreases and absorption increases when the CF4 concentration exceeds 0.03%. The decrease in the density of KrF(B) is dominantly the result of the interception of precursors to forming the upper laser level, as opposed to direct quenching.

Coulomb interactions between dust particles in plasma etching reactors

Summary form only given. We discuss results from a computer model for dust particle transport in reactive ion etching discharges where particle-particle Coulomb interactions are included. The basis of this study is a previously described dust transport simulation (DTS) which calculates particle trajectories by integrating the equations of motion due to the sum of the appropriate forces. Debye shielded Coulomb interactions between dust particles have been added to the DTS on a particle-by-particle basis. Results from the DTS indicate that particle-particle interactions modify trapping locations from localized points to extended sheets. Clouds or domes of particles typically observed above wafers are largely a consequence of particle-particle interactions which redistribute particles on nearly equipotential surfaces.