Viktoriia Postupalenko

A Universal Nucleoside with Strong Two-Band Switchable Fluorescence and Sensitivity to the Environment for Investigating DNA Interactions

With the aim of developing a new tool to investigate DNA interactions, a nucleoside analogue incorporating a 3-hydroxychromone (3HC) fluorophore as a nucleobase mimic was synthesized and incorporated into oligonucleotide chains. In comparison with existing fluorescent nucleoside analogues, this dye features exceptional environmental sensitivity switching between two well-resolved fluorescence bands. In labeled DNA, this nucleoside analogue does not alter the duplex conformation and exhibits a high fluorescence quantum yield. This probe is up to 50-fold brighter than 2-aminopurine, the fluorescent nucleoside standard. Moreover, the dual emission is highly sensitive to the polarity of the environment; thus, a strong shielding effect of the flanking bases from water was observed. With this nucleoside, the effect of a viral chaperone protein on DNA base stacking was site-selectively monitored.

Monitoring membrane binding and insertion of peptides by two-color fluorescent label

Herein, we developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. For this purpose, we labeled the N-terminus of three synthetic peptides, melittin, magainin 2 and poly-l-lysine capable to interact with lipid membranes. Binding of these peptides to lipid vesicles induced a strong fluorescence increase, which enabled to quantify the peptide-membrane interaction. Moreover, the dual emission of the label in these peptides correlated well with the depth of its insertion measured by the parallax quenching method. Thus, in melittin and magainin 2, which show deep insertion of their N-terminus, the label presented a dual emission corresponding to a low polar environment, while the environment of the poly-l-lysine N-terminus was rather polar, consistent with its location close to the bilayer surface. Using spectral deconvolution to distinguish the non-hydrated label species from the hydrated ones and two photon fluorescence microscopy to determine the probe orientation in giant vesicles, we found that the non-hydrated species were vertically oriented in the bilayer and constituted the best indicators for evaluating the depth of the peptide N-terminus in membranes. Thus, this label constitutes an interesting new tool for monitoring membrane binding and insertion of peptides.

Two-Color Fluorescent l-Amino Acid Mimic of Tryptophan for Probing Peptide-Nucleic Acid Complexes.

Non-natural amino acids are important tools for site-selective probing of peptide properties and interactions. Here, for the first time a fluorescent l-amino acid, exhibiting excited-state intramolecular proton transfer (ESIPT) and hydration-sensitive dual emission, was synthesized. It is an analogue of l-tryptophan bearing a slightly larger 2-(2-furyl)-3-hydroxychromone aromatic moiety instead of indole. This new amino acid was incorporated through solid-phase synthesis into NC(11-55), the zinc finger domain of the HIV-1 nucleocapsid protein, that exhibits potent nucleic acid chaperone properties. It was substituted for the Trp37 and Ala30 residues, located in the distal finger motif and the linker between the fingers of NC(11-55), respectively. Though the highly conserved Trp37 residue plays a key role in NC(11-55) structure and activity, its substitution for the new fluorescent analogue preserved the folding, the nucleic acid binding and chaperone activity of the peptide, indicating that the new amino acid can conservatively substitute Trp residues. In the presence of oligonucleotides, the Trp37-substituted peptide, but not the Ala30 variant, showed strong changes of the dual emission corresponding to local dehydration. The results are in line with NMR data, suggesting that the fluorescent amino acid interacts similarly to Trp37 with the nucleobases and is thus screened from water. Due to the exceptional sensitivity of its ESIPT fluorophore to hydration in highly polar environment, the new amino acid appears as a promising tool for substituting Trp residues and site-selectively investigating peptide-nucleic acid complexes.

Quantification of local hydration at the surface of biomolecules using dual-fluorescence labels.

By using four labels of the 3-hydroxyflavone family displaying selective sensitivity to hydrogen bond (HB) donors and poor response to other polar molecules, we developed an approach for measuring local water concentration [H(2)O](L) (or partial volume of water: W(A) = [H(2)O](L)/55.6) in the label surrounding both in solvent mixtures and in biomolecules by the intensity ratio of two emissive forms of the label, N*/T*. Using a series of binary water/solvent mixtures with limited preferential solvation effects, a linear dependence of log(N*/T*) on the local concentration of HB donor was obtained and then used as a calibration curve for estimating the W(A) values in the surroundings of the probes conjugated to biomolecules. By this approach, we estimated the hydration of the labels in different peptides and their complexes with DNAs. We found that W(A) values for the label at the peptide N-terminus are lower (0.63-0.91) than for free labels and depend strongly on the nature of the N-terminal amino acid. When complexed with different DNAs, the estimated hydration of the labels conjugated to the labeled peptides was much lower (W(A) = 0-0.47) and depended on the DNA nature and linker-label structure. Thus, the elaborated method allows a site-specific evaluation of hydration at the surface of a biomolecule through the determination of the partial volume of water. We believe the developed procedure can be successfully applied for monitoring hydration at the surface of any biomolecule or nanostructure.

Stimuli-Responsive Codelivery of Oligonucleotides and Drugs by Self-Assembled Peptide Nanoparticles

Ever more emerging combined treatments exploiting synergistic effects of drug combinations demand smart, responsive codelivery carriers to reveal their full potential. In this study, a multifunctional stimuli-responsive amphiphilic peptide was designed and synthesized to self-assemble into nanoparticles capable of co-bearing and -releasing hydrophobic drugs and antisense oligonucleotides for combined therapies. The rational design was based on a hydrophobic l-tryptophan-d-leucine repeating unit derived from a truncated sequence of gramicidin A (gT), to entrap hydrophobic cargo, which is combined with a hydrophilic moiety of histidines to provide electrostatic affinity to nucleotides. Stimuli-responsiveness was implemented by linking the hydrophobic and hydrophilic sequence through an artificial amino acid bearing a disulfide functional group (H3SSgT). Stimuli-responsive peptides self-assembled in spherical nanoparticles in sizes (100-200 nm) generally considered as preferable for drug delivery applications. Responsive peptide nanoparticles revealed notable nucleotide condensing abilities while maintaining the ability to load hydrophobic cargo. The disulfide cleavage site introduced in the peptide sequence induced responsiveness to physiological concentrations of reducing agent, serving to release the incorporated molecules. Furthermore, the peptide nanoparticles, singly loaded or coloaded with boron-dipyrromethene (BODIPY) and/or antisense oligonucleotides, were efficiently taken up by cells. Such amphiphilic peptides that led to noncytotoxic, reduction-responsive nanoparticles capable of codelivering hydrophobic and nucleic acid payloads simultaneously provide potential toward combined treatment strategies to exploit synergistic effects.

The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

ABSTRACT The HIV-1 Gag polyprotein precursor composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains orchestrates virus assembly via interactions between MA and the cell plasma membrane (PM) on one hand and NC and the genomic RNA on the other hand. As the Gag precursor can adopt a bent conformation, a potential interaction of the NC domain with the PM cannot be excluded during Gag assembly at the PM. To investigate the possible interaction of NC with lipid membranes in the absence of any interference from the other domains of Gag, we quantitatively characterized by fluorescence spectroscopy the binding of the mature NC protein to large unilamellar vesicles (LUVs) used as membrane models. We found that NC, either in its free form or bound to an oligonucleotide, was binding with high affinity (∼107 M−1) to negatively charged LUVs. The number of NC binding sites, but not the binding constant, was observed to decrease with the percentage of negatively charged lipids in the LUV composition, suggesting that NC and NC/oligonucleotide complexes were able to recruit negatively charged lipids to ensure optimal binding. However, in contrast to MA, NC did not exhibit a preference for phosphatidylinositol-(4,5)-bisphosphate. These results lead us to propose a modified Gag assembly model where the NC domain contributes to the initial binding of the bent form of Gag to the PM. IMPORTANCE The NC protein is a highly conserved nucleic acid binding protein that plays numerous key roles in HIV-1 replication. While accumulating evidence shows that NC either as a mature protein or as a domain of the Gag precursor also interacts with host proteins, only a few data are available on the possible interaction of NC with lipid membranes. Interestingly, during HIV-1 assembly, the Gag precursor is thought to adopt a bent conformation where the NC domain may interact with the plasma membrane. In this context, we quantitatively characterized the binding of NC, as a free protein or as a complex with nucleic acids, to lipid membranes and showed that the latter constitute a binding platform for NC. Taken together, our data suggest that the NC domain may play a role in the initial binding events of Gag to the plasma membrane during HIV-1 assembly.

Protein Delivery System Containing a Nickel‐Immobilized Polymer for Multimerization of Affinity‐Purified His‐Tagged Proteins Enhances Cytosolic Transfer

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.

Dual-fluorescence L-amino acid reports insertion and orientation of melittin peptide in cell membranes.

Monitoring insertion and orientation of peptides in situ on cell membranes remains a challenge. To this end, we synthesized an l-amino acid (AFaa) containing a dual-fluorescence dye of the 3-hydroxyflavone family, as a side chain. In contrast to other labeling approaches using a flexible linker, the AFaa fluorophore, introduced by solid phase synthesis into desired position of a peptide, is attached closely to its backbone with well-defined orientation, and, therefore, could reflect its localization in the membrane. This concept was validated by replacing the leucine-9 (L9) and tryptophan-19 (W19) residues by AFaa in melittin, a well-studied membrane-active peptide. Due to high sensitivity of AFaa dual emission to the environment polarity, we detected a much deeper insertion of L9 peptide position into the bilayer, compared to the W19 position. Moreover, using fluorescence microscopy with a polarized light excitation, we found different orientation of AFaa at L9 and W19 positions of melittin in the bilayers of giant vesicles and cellular membranes. These results suggested that in the natural membranes, similarly to the model lipid bilayers, melittin is preferentially oriented parallel to the membrane surface. The developed amino acid and the proposed methodology will be of interest to study other membrane peptides.

Intracellular delivery of functionally active proteins using self-assembling pyridylthiourea-polyethylenimine

Intracellular delivery of functionally active proteins into cells is emerging as a novel strategy for research and therapeutic applications. Here, we present the properties of a self-assembling pyridylthiourea-modified polyethylenimine (πPEI), which interacts with proteins and promotes their delivery into the cytosol of mammalian cells. In aqueous medium at pH7.4, self-association of πPEI in the presence of green fluorescent proteins (GFP) leads to supramolecular protein-entrapped assemblies. These assemblies protect GFP from losing its fluorescence upon pH variation and assist delivery/translocation into the cytosol of mammalian cells via the endocytic pathway. The scope of application of this delivery system was extended to antibodies against intracellular targets as illustrated using a monoclonal antibody directed against the HPV-16 viral E6 oncoprotein and an antibody directed against the threonine-927 phosporylation site of the EG5 kinesin spindle protein. The πPEI-mediated delivery of native anti-E6 antibodies or anti-E6 antibodies equipped with a nuclear localization signal (NLS), led to regeneration of the p53 tumor suppression protein in E6-transformed CaSki cells. Delivery of functionally active anti-EG5 antibodies, with the same polymer, reduced HeLa cell viability and appeared to perturb, as expected, chromosome segregation during mitosis. Altogether, these results provide an easy to use delivery system for extending the scope of application of antibodies for epitope recognition within living cells and may provide novel opportunities for selective interference of cell function by a steric hindrance modality.

Environmentally Sensitive Fluorescent Nucleoside Analogues for Surveying Dynamic Interconversions of Nucleic Acid Structures

Nucleic acids are characterized by a variety of dynamically interconverting structures that play a major role in transcriptional and translational regulation as well as recombination and repair. To monitor these interconversions, Förster resonance energy transfer (FRET)-based techniques can be used, but require two fluorophores that are typically large and can alter the DNA/RNA structure and protein binding. Additionally, events that do not alter the donor/acceptor distance and/or angular relationship are frequently left undetected. A more benign approach relies on fluorescent nucleobases that can substitute their native counterparts with minimal perturbation, such as the recently developed 2-thienyl-3-hydroxychromone (3HCnt) and thienoguanosine (th G). To demonstrate the potency of 3HCnt and th G in deciphering interconversion mechanisms, we used the conversion of the (-)DNA copy of the HIV-1 primer binding site (-)PBS stem-loop into (+)/(-)PBS duplex, as a model system. When incorporated into the (-)PBS loop, the two probes were found to be highly sensitive to the individual steps both in the absence and the presence of a nucleic acid chaperone, providing the first complete mechanistic description of this critical process in HIV-1 replication. The combination of the two distinct probes appears to be instrumental for characterizing structural transitions of nucleic acids under various stimuli.