Single-Molecule Spectroscopy of the Conjugated Polymer MEH-PPV

Single-Molecule Spectroscopy of the Conjugated Polymer MEH-PP

HIV-1 Nucleocapsid Protein Bends Double-Stranded Nucleic Acids

The human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein is believed to be unique among the nucleic acid (NA) binding proteins encoded by this retrovirus in being highly multifunctional and relatively nonsequence-specific. Underlying many of NC’s putative functions, including for example its chaperon-like activity for various steps of HIV-1 reverse transcription, is NC’s ability to partially melt short double-stranded regions of structured NAs, which is essentially a consequence of NC’s general binding preference for single-stranded bases. Herein we report a different, previously undiscovered, mode of NC/NA interaction, i.e., NC-induced sharp bending of short segments of fully duplexed DNA/DNA and DNA/RNA. We use single-molecule fluorescence resonance energy transfer (SM-FRET) in vitro to probe NC-induced NA bending and associated heterogeneous conformational dynamics for model NC/NA complexes. NC-induced NA bending may have important biological roles in the previously reported NC-mediated condensation of duplex proviral DNA in the HIV-1 life cycle

Single-Molecule Spectroscopic Study of Dynamic Nanoscale DNA Bending Behavior of HIV-1 Nucleocapsid Protein

We have studied the conformational dynamics associated with the nanoscale DNA bending induced by human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein using single-molecule Förster resonance energy transfer (SM-FRET). To gain molecular-level insights into how the HIV-1 NC locally distorts the structures of duplexed DNA segments, the dynamics, reversibility, and sequence specificity of the DNA bending behavior of NC have been systematically studied. We have performed SM-FRET measurements on a series of duplexed DNA segments with varying sequences, lengths, and local structures in the presence of the wide-type HIV-1 NC and NC mutants lacking either the basic N-terminal domain or the zinc fingers. On the basis of the SM-FRET results, we have proposed a possible mechanism for the NC-induced DNA bending in which both NC’s zinc fingers and N-terminal domain are found to play crucial roles. The SM-FRET results reported here add new mechanistic insights into the biological behaviors and functions of HIV-1 NC as a retroviral DNA-architectural protein which may play critical roles in the compaction, nuclear import, and integration of the proviral DNA during the retroviral life cycle

Single-Molecule Spectroelectrochemistry (SMS-EC)

We introduce single-molecule spectroelectrochemistry (SMS-EC), a powerful new technique for studying electrochemical kinetics in highly heterogeneous systems. This technique uses fluorescence single-molecule spectroscopy to indirectly measure electrochemical kinetics one molecule at a time, offering for the first time the distribution of key electrochemical variables, such as the half-wave potential, E1/2, not just the ensemble averages. In SMS-EC, the potential of the working electrode of an electrochemical cell is linearly scanned while simultaneously measuring the florescence intensity, Ifl(t), of individual single molecules as a function of time in a wide-field microscope. SMS-EC is used herein to study the oxidation at an indium tin oxide (ITO) electrode of single molecules of the organic conjugated polymer F8BT. The results reveal both excited singlet state and ground state oxidation of F8BT. The latter process occurs over a narrow distribution of single-molecule half-wave potential values, indicating a relatively uniform electrochemical potential at the electrode

NSOM Investigations of the Spectroscopy and Morphology of Self-Assembled Multilayered Thin Films

Near-field scanning optical microscopy (NSOM) and atomic force microscopy (AFM) have been employed to spatially resolve the complex nanoscale morphologies, spectroscopy, and energy-transfer efficiencies of self-assembled multilayered structures composed of alternating layers of α-zirconium phosphate [α-Zr(HPO4)2] (ZrP) and dye-labeled poly(allylamine hydrochloride) (dye-PAH) (where dye = Fluorescein (FL), Rhodamine B (RhB), or Texas Red (TR)). Two types of multilayer films have been investigated, namely, glass/anchor/ZrP/dye-PAH and glass/anchor/ZrP/dye-PAH/ZrP/dye-PAH, which were formed by the sequential layer-by-layer adsorption of the charged polyelectrolyte component layers. High- and low-coverage films were investigated. The glass/anchor/ZrP assemblies were shown to consist of a densely packed “tiled” motif of ZrP sheets which lie flat on the surface and cover more than 95% of the area, with average plate sizes of height = 13 (7) Å, width ≈ 150 nm. The dye-labeled polymer layers in glass/anchor/ZrP/dye-PAH and glass/anchor/ZrP/dye-PAH/ZrP/dye-PAH were shown to adhere to the surface of the ZrP sheets and fill in the cracks between the sheets to a lesser extent. The measured heights of these polymer-coated multilayer films are 26(9) and 48(15) Å, respectively. These heights are consistent with theoretical estimates of ideally packed ionic films (28 and 48 Å, respectively). Dual-wavelength fluorescence NSOM imaging at 580 nm and >610 nm and near-field photobleach experiments were used to spatially resolve nanoscopic regions that display energy transfer between the layers

NSOM Investigations of the Spectroscopy and Morphology of Self-Assembled Multilayered Thin Films

Near-field scanning optical microscopy (NSOM) and atomic force microscopy (AFM) have been employed to spatially resolve the complex nanoscale morphologies, spectroscopy, and energy-transfer efficiencies of self-assembled multilayered structures composed of alternating layers of α-zirconium phosphate [α-Zr(HPO4)2] (ZrP) and dye-labeled poly(allylamine hydrochloride) (dye-PAH) (where dye = Fluorescein (FL), Rhodamine B (RhB), or Texas Red (TR)). Two types of multilayer films have been investigated, namely, glass/anchor/ZrP/dye-PAH and glass/anchor/ZrP/dye-PAH/ZrP/dye-PAH, which were formed by the sequential layer-by-layer adsorption of the charged polyelectrolyte component layers. High- and low-coverage films were investigated. The glass/anchor/ZrP assemblies were shown to consist of a densely packed “tiled” motif of ZrP sheets which lie flat on the surface and cover more than 95% of the area, with average plate sizes of height = 13 (7) Å, width ≈ 150 nm. The dye-labeled polymer layers in glass/anchor/ZrP/dye-PAH and glass/anchor/ZrP/dye-PAH/ZrP/dye-PAH were shown to adhere to the surface of the ZrP sheets and fill in the cracks between the sheets to a lesser extent. The measured heights of these polymer-coated multilayer films are 26(9) and 48(15) Å, respectively. These heights are consistent with theoretical estimates of ideally packed ionic films (28 and 48 Å, respectively). Dual-wavelength fluorescence NSOM imaging at 580 nm and >610 nm and near-field photobleach experiments were used to spatially resolve nanoscopic regions that display energy transfer between the layers

Single-Molecule Study of the Inhibition of HIV-1 Transactivation Response Region DNA/DNA Annealing by Argininamide

Single-molecule spectroscopy was used to examine how a model inhibitor of HIV-1, argininamide, modulates the nucleic acid chaperone activity of the nucleocapsid protein (NC) in the minus-strand transfer step of HIV-1 reverse transcription, in vitro. In minus-strand transfer, the transactivation response region (TAR) RNA of the genome is annealed to the complementary “TAR DNA” generated during minus-strand strong-stop DNA synthesis. Argininamide and its analogs are known to bind to the hairpin bulge region of TAR RNA as well as to various DNA loop structures, but its ability to inhibit the strand transfer process has only been implied. Here, we explore how argininamide modulates the annealing kinetics and secondary structure of TAR DNA. The studies reveal that the argininamide inhibitory mechanism involves a shift of the secondary structure of TAR, away from the NC-induced “Y” form, an intermediate in reverse transcription, and toward the free closed or “C” form. In addition, more potent inhibition of the loop-mediated annealing pathway than stem-mediated annealing is observed. Taken together, these data suggest a molecular mechanism wherein argininamide inhibits NC-facilitated TAR RNA/DNA annealing in vitro by interfering with the formation of key annealing intermediates

NSOM Investigations of the Spectroscopy and Morphology of Self-Assembled Multilayered Thin Films

Near-field scanning optical microscopy (NSOM) and atomic force microscopy (AFM) have been employed to spatially resolve the complex nanoscale morphologies, spectroscopy, and energy-transfer efficiencies of self-assembled multilayered structures composed of alternating layers of α-zirconium phosphate [α-Zr(HPO4)2] (ZrP) and dye-labeled poly(allylamine hydrochloride) (dye-PAH) (where dye = Fluorescein (FL), Rhodamine B (RhB), or Texas Red (TR)). Two types of multilayer films have been investigated, namely, glass/anchor/ZrP/dye-PAH and glass/anchor/ZrP/dye-PAH/ZrP/dye-PAH, which were formed by the sequential layer-by-layer adsorption of the charged polyelectrolyte component layers. High- and low-coverage films were investigated. The glass/anchor/ZrP assemblies were shown to consist of a densely packed “tiled” motif of ZrP sheets which lie flat on the surface and cover more than 95% of the area, with average plate sizes of height = 13 (7) Å, width ≈ 150 nm. The dye-labeled polymer layers in glass/anchor/ZrP/dye-PAH and glass/anchor/ZrP/dye-PAH/ZrP/dye-PAH were shown to adhere to the surface of the ZrP sheets and fill in the cracks between the sheets to a lesser extent. The measured heights of these polymer-coated multilayer films are 26(9) and 48(15) Å, respectively. These heights are consistent with theoretical estimates of ideally packed ionic films (28 and 48 Å, respectively). Dual-wavelength fluorescence NSOM imaging at 580 nm and >610 nm and near-field photobleach experiments were used to spatially resolve nanoscopic regions that display energy transfer between the layers

NSOM Investigations of the Spectroscopy and Morphology of Self-Assembled Multilayered Thin Films

Near-field scanning optical microscopy (NSOM) and atomic force microscopy (AFM) have been employed to spatially resolve the complex nanoscale morphologies, spectroscopy, and energy-transfer efficiencies of self-assembled multilayered structures composed of alternating layers of α-zirconium phosphate [α-Zr(HPO4)2] (ZrP) and dye-labeled poly(allylamine hydrochloride) (dye-PAH) (where dye = Fluorescein (FL), Rhodamine B (RhB), or Texas Red (TR)). Two types of multilayer films have been investigated, namely, glass/anchor/ZrP/dye-PAH and glass/anchor/ZrP/dye-PAH/ZrP/dye-PAH, which were formed by the sequential layer-by-layer adsorption of the charged polyelectrolyte component layers. High- and low-coverage films were investigated. The glass/anchor/ZrP assemblies were shown to consist of a densely packed “tiled” motif of ZrP sheets which lie flat on the surface and cover more than 95% of the area, with average plate sizes of height = 13 (7) Å, width ≈ 150 nm. The dye-labeled polymer layers in glass/anchor/ZrP/dye-PAH and glass/anchor/ZrP/dye-PAH/ZrP/dye-PAH were shown to adhere to the surface of the ZrP sheets and fill in the cracks between the sheets to a lesser extent. The measured heights of these polymer-coated multilayer films are 26(9) and 48(15) Å, respectively. These heights are consistent with theoretical estimates of ideally packed ionic films (28 and 48 Å, respectively). Dual-wavelength fluorescence NSOM imaging at 580 nm and >610 nm and near-field photobleach experiments were used to spatially resolve nanoscopic regions that display energy transfer between the layers

NSOM Investigations of the Spectroscopy and Morphology of Self-Assembled Multilayered Thin Films

Near-field scanning optical microscopy (NSOM) and atomic force microscopy (AFM) have been employed to spatially resolve the complex nanoscale morphologies, spectroscopy, and energy-transfer efficiencies of self-assembled multilayered structures composed of alternating layers of α-zirconium phosphate [α-Zr(HPO4)2] (ZrP) and dye-labeled poly(allylamine hydrochloride) (dye-PAH) (where dye = Fluorescein (FL), Rhodamine B (RhB), or Texas Red (TR)). Two types of multilayer films have been investigated, namely, glass/anchor/ZrP/dye-PAH and glass/anchor/ZrP/dye-PAH/ZrP/dye-PAH, which were formed by the sequential layer-by-layer adsorption of the charged polyelectrolyte component layers. High- and low-coverage films were investigated. The glass/anchor/ZrP assemblies were shown to consist of a densely packed “tiled” motif of ZrP sheets which lie flat on the surface and cover more than 95% of the area, with average plate sizes of height = 13 (7) Å, width ≈ 150 nm. The dye-labeled polymer layers in glass/anchor/ZrP/dye-PAH and glass/anchor/ZrP/dye-PAH/ZrP/dye-PAH were shown to adhere to the surface of the ZrP sheets and fill in the cracks between the sheets to a lesser extent. The measured heights of these polymer-coated multilayer films are 26(9) and 48(15) Å, respectively. These heights are consistent with theoretical estimates of ideally packed ionic films (28 and 48 Å, respectively). Dual-wavelength fluorescence NSOM imaging at 580 nm and >610 nm and near-field photobleach experiments were used to spatially resolve nanoscopic regions that display energy transfer between the layers