Exploiting Conjugated Polyelectrolyte Photophysics toward Monitoring Real-Time Lipid Membrane-Surface Interaction Dynamics at the Single-Particle Level

Herein we report the real-time observation of the interaction dynamics between cationic liposomes flowing in solution and a surface-immobilized charged scaffolding formed by the deposition of conjugated polyanion poly­[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene (MPS-PPV) onto 100-nm-diameter SiO₂ nanoparticles (NPs). Contact of the freely floating liposomes with the polymer-coated surfaces led to the formation of supported lipid bilayers (SLBs). The interaction of the incoming liposomes with MPS-PPV adsorbed on individual SiO₂ nanoparticles promoted the deaggregation of the polymer conformation and led to large emission intensity enhancements. Single-particle total internal reflection fluorescence microscopy studies exploited this phenomenon as a way to monitor the deformation dynamics of liposomes on surface-immobilized NPs. The MPS-PPV emission enhancement (up to 25-fold) reflected on the extent of membrane contact with the surface of the NP and was correlated with the size of the incoming liposome. The time required for the MPS-PPV emission to reach a maximum (ranging from 400 to 1000 ms) revealed the dynamics of membrane deformation and was also correlated with the liposome size. Cryo-TEM experiments complemented these results by yielding a structural view of the process. Immediately following the mixing of liposomes and NPs the majority of NPs had one or more adsorbed liposomes, yet the presence of a fully formed SLB was rare. Prolonged incubation of liposomes and NPs showed completely formed SLBs on all of the NPs, confirming that the liposomes eventually ruptured to form SLBs. We foresee that the single-particle studies we report herein may be readily extended to study membrane dynamics of other lipids including cellular membranes in live cell studies and to monitor the formation of polymer-cushioned SLBs

Enhancing the Emissive Properties of Poly(<i>p</i>-phenylenevinylene)-Conjugated Polyelectrolyte-Coated SiO₂ Nanoparticles

Here we describe single-particle imaging studies conducted on the conjugated polyelecrolyte poly­[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene] (MPS-PPV) supported on SiO2 nanoparticles. The particles are subjected to a time-programmed sequence involving addition and removal of different additives, including excited-triplet-state quenchers and scavengers of singlet oxygen as well as ground-state oxygen. Our studies show that these additives enhance the emission intensity and photostability of the nanoparticles and may further repair photodamaged conjugated polymer. The ability to monitor the emission from individual particles along multiple cycles under a range of conditions provides a mechanistic insight into the action of these additives

Enhancing the Emissive Properties of Poly(<i>p</i>-phenylenevinylene)-Conjugated Polyelectrolyte-Coated SiO₂ Nanoparticles

Here we describe single-particle imaging studies conducted on the conjugated polyelecrolyte poly­[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene] (MPS-PPV) supported on SiO₂ nanoparticles. The particles are subjected to a time-programmed sequence involving addition and removal of different additives, including excited-triplet-state quenchers and scavengers of singlet oxygen as well as ground-state oxygen. Our studies show that these additives enhance the emission intensity and photostability of the nanoparticles and may further repair photodamaged conjugated polymer. The ability to monitor the emission from individual particles along multiple cycles under a range of conditions provides a mechanistic insight into the action of these additives

Dye Lipophilicity and Retention in Lipid Membranes: Implications for Single-Molecule Spectroscopy

Fluorescence studies of individual lipid vesicles rely on the proper positioning of probes in the lipid milieu. This is true for both positional tags and chemoselective fluorogenic probes that undergo chemical modification following reaction with an analyte of interest within the lipid environment. The present report describes lipophilicity and localization estimations for a series of BODIPY dyes bearing substituents of varying hydrophobicity. We also studied fluorogenic trap–reporter probes that undergo fluorescence emission enhancement upon trapping of reactive oxygen species (ROS), including lipid peroxyl radicals. We show that caution has to be taken to extrapolate ensemble partition measurements of dyes to the single-molecule regime as a result of the dramatically different lipid concentration prevailing in ensemble versus single-molecule experiments. We show that the mole fraction of dyes that remains embedded in liposomes during a typical single-molecule experiment may be accurately determined from a ratiometric single-particle imaging analysis. We further demonstrate that fluorescence correlation spectroscopy (FCS) provides a very rapid and reliable estimate of the lipophilic nature of a given dye under highly dilute single-molecule-like conditions. Our combined single-particle spectroscopy and FCS experiments suggest that the minimal mole fraction of membrane-associated dyes (<i>x</i>_m) as determined from FCS experiments is about 0.5 for adequate dye retention during single-molecule imaging in lipid membranes. Our work further highlights the dramatic effect that chemical modifications can have on chemoselective fluorogenic probe localization

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

Binding Kinetics and Affinities of Heterodimeric versus Homodimeric HIV‑1 Reverse Transcriptase on DNA–DNA Substrates at the Single-Molecule Level

During viral replication, HIV-1 reverse transcriptase (RT) plays a pivotal role in converting genomic RNA into proviral DNA. While the biologically relevant form of RT is the p66–p51 heterodimer, two recombinant homodimer forms of RT, p66–p66 and p51–p51, are also catalytically active. Here we investigate the binding of the three RT isoforms to a fluorescently labeled 19/50-nucleotide primer/template DNA duplex by exploiting single-molecule protein-induced fluorescence enhancement (SM-PIFE). PIFE, which does not require labeling of the protein, allows us to directly visualize the binding/unbinding of RT to a double-stranded DNA substrate. We provide values for the association and dissociation rate constants of the RT homodimers p66–p66 and p51–p51 with a double-stranded DNA substrate and compare those to the values recorded for the RT heterodimer p66–p51. We also report values for the equilibrium dissociation constant for the three isoforms. Our data reveal great similarities in the intrinsic binding affinities of p66–p51 and p66–p66, with characteristic <i>K</i>_d values in the nanomolar range, much smaller (50–100-fold) than that of p51–p51. Our data also show discrepancies in the association/dissociation dynamics among the three dimeric RT isoforms. Our results further show that the apparent binding affinity of p51–p51 for its DNA substrate is to a great extent time-dependent when compared to that of p66–p66 and p66–p51, and is more likely determined by the dimer dissociation into its constituent monomers rather than the intrinsic binding affinity of dimeric RT

Dynamic Interconversions of HCV Helicase Binding Modes on the Nucleic Acid Substrate

The dynamics involved in the interaction between hepatitis C virus nonstructural protein 3 (NS3) C-terminal helicase and its nucleic acid substrate have been the subject of interest for some time given the key role of this enzyme in viral replication. Here, we employed fluorescence-based techniques and focused on events that precede the unwinding process. Both ensemble Förster resonance energy transfer (FRET) and ensemble protein induced fluorescence enhancement (PIFE) assays show binding on the 3′ single-stranded overhang of model DNA substrates (>5 nucleotides) with no preference for the single-stranded/double-stranded (ss/ds) junction. Single-molecule PIFE experiments revealed three enhancement levels that correspond to three discrete binding sites at adjacent bases. The enzyme is able to transition between binding sites in both directions without dissociating from the nucleic acid. In contrast, the NS3 mutant W501A, which is unable to engage in stacking interactions with the DNA, is severely compromised in this switching activity. Altogether our data are consistent with a model for NS3 dynamics that favors ATP-independent random binding and sliding by one and two nucleotides along the overhang of the loading strand

Interactions of the Disordered Domain II of Hepatitis C Virus NS5A with Cyclophilin A, NS5B, and Viral RNA Show Extensive Overlap

Domain II of the nonstructural protein 5 (NS5A) of the hepatitis C virus (HCV) is involved in intermolecular interactions with the viral RNA genome, the RNA-dependent RNA polymerase NS5B, and the host factor cyclophilin A (CypA). However, domain II of NS5A (NS5A^DII) is largely disordered, which makes it difficult to characterize the protein–protein or protein–nucleic acid interfaces. Here we utilized a mass spectrometry-based protein footprinting approach in attempts to characterize regions forming contacts between NS5A^DII and its binding partners. In particular, we compared surface topologies of lysine and arginine residues in the context of free and bound NS5A^DII. These experiments have led to the identification of an RNA binding motif (³⁰⁵RSR­KFPR³¹¹) in an arginine-rich region of NS5A^DII. Furthermore, we show that K308 is indispensable for both RNA and NS5B binding, whereas W316, further downstream, is essential for protein–protein interactions with CypA and NS5B. Most importantly, NS5A^DII binding to NS5B involves a region associated with RNA binding within NS5B. This interaction down-regulated RNA synthesis by NS5B, suggesting that NS5A^DII modulates the activity of NS5B and potentially regulates HCV replication

Multiple Hydrogen Bonds Tuning Guest/Host Excited-State Proton Transfer Reaction: Its Application in Molecular Recognition

A molecular recognition concept exploiting multiple-hydrogen-bond fine-tuned excited-state proton-transfer (ESPT) was conveyed using 3,4,5,6-tetrahydrobis(pyrido[3,2-g]indolo)[2,3-a:3‘,2‘-j]acridine (1a). The catalytic type 1a/carboxylic acids hydrogen-bonding (HB) complexes undergo ultrafast ESPT, resulting in an anomalously large Stokes shifted tautomer emission (λmax ≈ 600 nm). Albeit forming a quadruple HB complex, ESPT is prohibited in the noncatalytic-type 1a/urea complexes (λmax ≈ 430 nm). The HB configuration tuning ESPT properties lead to a feasible design for sensing multiple-HB-site analytes of biological interest

Multiple Hydrogen Bonds Tuning Guest/Host Excited-State Proton Transfer Reaction: Its Application in Molecular Recognition

A molecular recognition concept exploiting multiple-hydrogen-bond fine-tuned excited-state proton-transfer (ESPT) was conveyed using 3,4,5,6-tetrahydrobis(pyrido[3,2-g]indolo)[2,3-a:3‘,2‘-j]acridine (1a). The catalytic type 1a/carboxylic acids hydrogen-bonding (HB) complexes undergo ultrafast ESPT, resulting in an anomalously large Stokes shifted tautomer emission (λmax ≈ 600 nm). Albeit forming a quadruple HB complex, ESPT is prohibited in the noncatalytic-type 1a/urea complexes (λmax ≈ 430 nm). The HB configuration tuning ESPT properties lead to a feasible design for sensing multiple-HB-site analytes of biological interest