Iron Oxide Nanoparticles Functionalized with Fucoidan: a Potential Theranostic Nanotool for Hepatocellular Carcinoma.

Fucoidan is a natural sulfated polysaccharide with a large range of biological activities including anticancer and anti-oxidation activities. Hepatocellular carcinoma is the fourth most common aggressive cancer type. The aim of this study was to investigate the bioactivity of free fucoidan versus its vectorization using nanoparticles (NPs) in human hepatoma cells, Huh-7. Iron oxide NPs were functionalized with fucoidan by a one-step surface complexation. NP cellular uptake was quantified by magnetic measurement at various extracellular iron concentrations. Cell invasion and migration were reduced with NPs while free fucoidan increases these events at low fucoidan concentration (≤0.5 µM). Concomitantly, a high decrease of reactive oxygen species production related with a decrease of the matrix metalloproteinase-9 activity and an increase of its expression was observed with NPs compared to free fucoidan. A proteomic analysis evidenced that some fucoidan regulated proteins appeared, which were related to protein synthesis, N-glycan processing, and cellular stress. To our knowledge, this is the first study which reveals such activity induced by fucoidan. These results pave the way for USPIO-fucoidan-NPs as potential theranostic nanotools for hepatocellular carcinoma treatment.

Amyloid-like Hfq interaction with single-stranded DNA: involvement in recombination and replication in Escherichia coli.

Interactions between proteins and single-stranded DNA (ssDNA) are crucial for many fundamental biological processes, including DNA replication and genetic recombination. Thus, understanding detailed mechanisms of these interactions is necessary to uncover regulatory rules occurring in all living cells. The RNA-binding Hfq is a pleiotropic bacterial regulator that mediates many aspects of nucleic acid metabolism. The protein notably mediates mRNA stability and translation efficiency by using stress-related small regulatory RNA as cofactors. In addition, Hfq helps to compact double-stranded DNA. In this paper, we focused on the action of Hfq on ssDNA. A combination of experimental methodologies, including spectroscopy and molecular imaging, has been used to probe the interactions of Hfq and its amyloid C-terminal region with ssDNA. Our analysis revealed that Hfq binds to ssDNA. Moreover, we demonstrate for the first time that Hfq drastically changes the structure and helical parameters of ssDNA, mainly due to its C-terminal amyloid-like domain. The formation of the nucleoprotein complexes between Hfq and ssDNA unveils important implications for DNA replication and recombination.

SRCD and FTIR Spectroscopies to Monitor Protein-Induced Nucleic Acid Remodeling.

Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies are fast techniques providing important information about the conformation of nucleic acids and proteins. These vibrational and electronic absorption spectroscopies are extremely sensitive to any change in molecular structure. While numerous reviews describe how to analyze DNA structure alone or in the presence of proteins using FTIR and CD, analyses of RNA are scarce. Nevertheless, RNA remodeling proteins are important factors involved in a multitude of roles in the cell. In this chapter, we present applications of synchrotron radiation circular dichroism (SRCD) and FTIR to analyze how proteins may change RNA structure. These include the analysis of RNA melting, or stabilization, of change in helical parameters and base stacking. The effects on the structure of RNA remodeling proteins are also presented.

Application of FTIR Spectroscopy to Analyze RNA Structure.

Fourier transform infrared (FTIR) spectroscopy has been widely used for the analysis of both protein and nucleic acid secondary structure. This is one of the vibration spectroscopy methods that are extremely sensitive to any change in molecular structure. While numerous reports describe how to proceed to analyze protein and deoxyribonucleic acid (DNA) structures using FTIR, reports related to the analyses of ribonucleic acids (RNAs) are few. Nevertheless, RNAs are versatile molecules involved in a multitude of roles in the cell. In this chapter, we present applications of FTIR for the structural analysis of RNA, including the analysis of helical parameters and noncanonical base pairing, often found in RNA. The effect of temperature pretreatment, which has a great impact on RNA folding, will also be discussed.

Role of Hfq in Genome Evolution: Instability of G-Quadruplex Sequences in E. coli.

Certain G-rich DNA repeats can form quadruplex in bacterial chromatin that can present blocks to DNA replication and, if not properly resolved, may lead to mutations. To understand the participation of quadruplex DNA in genomic instability in Escherichia coli (E. coli), mutation rates were measured for quadruplex-forming DNA repeats, including (G3T)4, (G3T)8, and a RET oncogene sequence, cloned as the template or nontemplate strand. We evidence that these alternative structures strongly influence mutagenesis rates. Precisely, our results suggest that G-quadruplexes form in E. coli cells, especially during transcription when the G-rich strand can be displaced by R-loop formation. Structure formation may then facilitate replication misalignment, presumably associated with replication fork blockage, promoting genomic instability. Furthermore, our results also evidence that the nucleoid-associated protein Hfq is involved in the genetic instability associated with these sequences. Hfq binds and stabilizes G-quadruplex structure in vitro and likely in cells. Collectively, our results thus implicate quadruplexes structures and Hfq nucleoid protein in the potential for genetic change that may drive evolution or alterations of bacterial gene expression.

Bimodal Fucoidan-Coated Zinc Oxide/Iron Oxide-Based Nanoparticles for the Imaging of Atherothrombosis.

A polyol method was used to obtain ultrasmall ZnO nanoparticles (NPs) doped with iron ions and coated with a low molecular weight fucoidan in order to perform in vivo MR and ex vivo fluorescence imaging of athrothrombosis. During the synthesis, the early elimination of water by azeotropic distillation with toluene allowed us to produce NPs which size, determined by XRD and TEM, decreased from 7 nm to 4 nm with the increase of iron/zinc ratios from 0.05 to 0.50 respectively. For the highest iron content (NP-0.50) NPs were evidenced as a mixture of nanocrystals made of wurtzite and cubic phase with a molar ratio of 2.57:1, although it was not possible to distinguish one from the other by TEM. NP-0.50 were superparamagnetic and exhibited a large emission spectrum at 470 nm when excited at 370 nm. After surface functionalization of NP-0.50 with fucoidan (fuco-0.50), the hydrodynamic size in the physiological medium was 162.0 ± 0.4 nm, with a corresponding negative zeta potential of -48.7 ± 0.4 mV, respectively. The coating was evidenced by FT-IR spectra and thermogravimetric analysis. Aqueous suspensions of fuco-0.50 revealed high transverse proton relaxivities (T2) with an r2 value of 173.5 mM-1 s-1 (300 K, 7.0 T) and remained stable for more than 3 months in water or in phosphate buffer saline without evolution of the hydrodynamic size and size distribution. No cytotoxic effect was observed on human endothelial cells up to 48 h with these NPs at a dose of 0.1 mg/mL. After injection into a rat model of atherothrombosis, MR imaging allowed the localization of diseased areas and the subsequent fluorescence imaging of thrombus on tissue slices.

Revised role for Hfq bacterial regulator on DNA topology.

Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNA. Besides these RNA-related functions, Hfq has also been described as one of the nucleoid associated proteins shaping the bacterial chromosome. Therefore, Hfq appears as a versatile nucleic acid-binding protein, which functions are probably even more numerous than those initially suggested. For instance, E. coli Hfq, and more precisely its C-terminal region (CTR), has been shown to induce DNA compaction into a condensed form. In this paper, we establish that DNA induces Hfq-CTR amyloidogenesis, resulting in a change of DNA local conformation. Furthermore, we clarify the effect of Hfq on DNA topology. Our results evidence that, even if the protein has a strong propensity to compact DNA thanks to its amyloid region, it does not affect overall DNA topology. We confirm however that hfq gene disruption influences plasmid supercoiling in vivo, indicating that the effect on DNA topology in former reports was indirect. Most likely, this effect is related to small regulatory sRNA-Hfq-based regulation of another protein that influences DNA supercoiling, possibly a nucleoid associated protein such as H-NS or Dps. Finally, we hypothesise that this indirect effect on DNA topology explains, at least partially, the previously reported effect of Hfq on plasmid replication efficiency.

Synthetic melanin bound to subunit vaccine antigens significantly enhances CD8+ T-cell responses.

Cytotoxic T-lymphocytes (CTLs) play a key role in immunity against cancer; however, the induction of CTL responses with currently available vaccines remains difficult. Because several reports have suggested that pigmentation and immunity might be functionally linked, we investigated whether melanin can act as an adjuvant in vaccines. Short synthetic peptides (8-35 amino acids long) containing T-cell epitopes were mixed with a solution of L-Dopa, a precursor of melanin. The mixture was then oxidized to generate nanoparticles of melanin-bound peptides. Immunization with melanin-bound peptides efficiently triggered CTL responses in mice, even against self-antigens and at a very low dose of peptides (microgram range). Immunization against a tumor antigen inhibited the growth of established tumors in mice, an effect that was abrogated by the depletion of CD8+ lymphocytes. These results demonstrate the efficacy of melanin as a vaccine adjuvant.

Identification of a Pro-Angiogenic Potential and Cellular Uptake Mechanism of a LMW Highly Sulfated Fraction of Fucoidan from Ascophyllum nodosum.

Herein we investigate the structure/function relationships of fucoidans from Ascophyllum nodosum to analyze their pro-angiogenic effect and cellular uptake in native and glycosaminoglycan-free (GAG-free) human endothelial cells (HUVECs). Fucoidans are marine sulfated polysaccharides, which act as glycosaminoglycans mimetics. We hypothesized that the size and sulfation rate of fucoidans influence their ability to induce pro-angiogenic processes independently of GAGs. We collected two fractions of fucoidans, Low and Medium Molecular Weight Fucoidan (LMWF and MMWF, respectively) by size exclusion chromatography and characterized their composition (sulfate, fucose and uronic acid) by colorimetric measurement and Raman and FT-IR spectroscopy. The high affinities of fractionated fucoidans to heparin binding proteins were confirmed by Surface Plasmon Resonance. We evidenced that LMWF has a higher pro-angiogenic (2D-angiogenesis on Matrigel) and pro-migratory (Boyden chamber) potential on HUVECs, compared to MMWF. Interestingly, in a GAG-free HUVECs model, LMWF kept a pro-angiogenic potential. Finally, to evaluate the association of LMWF-induced biological effects and its cellular uptake, we analyzed by confocal microscopy the GAGs involvement in the internalization of a fluorescent LMWF. The fluorescent LMWF was mainly internalized through HUVEC clathrin-dependent endocytosis in which GAGs were partially involved. In conclusion, a better characterization of the relationships between the fucoidan structure and its pro-angiogenic potential in GAG-free endothelial cells was required to identify an adapted fucoidan to enhance vascular repair in ischemia.

Vectorization of Nucleic Acids for Therapeutic Approach: Tutorial Review.

Oligonucleotides present a high therapeutic potential for a wide variety of diseases. However, their clinical development is limited by their degradation by nucleases and their poor blood circulation time. Depending on the administration mode and the cellular target, these macromolecules will have to cross the vascular endothelium, to diffuse through the extracellular matrix, to be transported through the cell membrane, and finally to reach the cytoplasm. To overcome these physiological barriers, many strategies have been developed. Here, we review different methods of DNA vectorization, discuss limitations and advantages of the various vectors, and provide new perspectives for future development.

Iron Oxide Nanoparticles Coated with a Phosphorothioate Oligonucleotide and a Cationic Peptide: Exploring Four Different Ways of Surface Functionalization.

The superparamagnetic iron oxide nanoparticles (SPIONs) have great potential in therapeutic and diagnostic applications. Due to their superparamagnetic behavior, they are used clinically as a Magnetic Resonance Imaging (MRI) contrast agent. Iron oxide nanoparticles are also recognized todays as smart drug-delivery systems. However, to increase their specificity, it is essential to functionalize them with a molecule that effectively targets a specific area of the body. Among the molecules that can fulfill this role, peptides are excellent candidates. Oligonucleotides are recognized as potential drugs for various diseases but suffer from poor uptake and intracellular degradation. In this work, we explore four different strategies, based on the electrostatic interactions between the different partners, to functionalize the surface of SPIONs with a phosphorothioate oligonucleotide (ODN) and a cationic peptide labeled with a fluorophore. The internalization of the nanoparticles has been evaluated in vitro on RAW 264.7 cells. Among these strategies, the "«one-step assembly¼", i.e., the direct complexation of oligonucleotides and peptides on iron oxide nanoparticles, provides the best way of coating for the internalization of the nanocomplexes.

Easily controlled grafting of oligonucleotides on γFe2O3 nanoparticles: physicochemical characterization of DNA organization and biological activity studies.

We report a one-step process to functionalize superparamagnetic iron oxide nanoparticle (SPIO-NP) surfaces with a controlled number of oligonucleotides. For this study, we use a specific oligonucleotide targeting the signal transducer and activator of transcription 3 (STAT3), a key regulator of cell survival and proliferation. This oligonucleotide is self-complementary and can adopt a hairpin structure. It is labeled with the fluorescein amidite group at the 3'-end. The polyanionic DNA is electrostatically attracted onto the positively charged surface of the bare SPIO-NPs. During synthesis, the molar ratio between the oligonucleotides and nanoparticles was varied from 17.5 to 175. For particles with a mean diameter of 10 nm, a nanoparticle surface saturation is observed corresponding to 70 DNA strands per particle. The increase of DNA density per nanoparticle is correlated to a transition from the hairpin structure adsorbed horizontally on the nanoparticle surface to a vertically ordered surface packing assembly. An in vitro study on human colon carcinoma cell line SW480 shows that the kinetics of internalization and biological activity of the NPs seem to be dependent on the oligonucleotide density. Cell death and the kinetics of internalization are favored by a low density of oligonucleotides.

Thermodynamic aspects of the self-assembly of DsrA, a small noncoding RNA from Escherichia coli.

DsrA is an Escherichia coli small noncoding RNA that acts by base pairing to some mRNAs in order to control their translation and turnover. It was recently shown that DsrA is able to self-associate in a way similar to DNA and to build nanostructures. Although functional consequence of this RNA self-assembly in vivo is not yet understood, the formation of such an assemblage more than likely influences the noncoding RNA function. We report here for the first time the thermodynamic basis of this natural RNA self-assembly. In particular we show that assembling of the ribonucleic acid is enthalpy driven and that the versatility of the RNA molecule is important for the polymerisation; indeed, an equivalent DNA sequence is unable to make a nanoassembly. The origin of the difference is discussed herein.

Electrostatic assembly of a DNA superparamagnetic nano-tool for simultaneous intracellular delivery and in situ monitoring.

A superparamagnetic γFe(2)O(3) nanocarrier was developed, characterized by spectroscopic methods and evaluated for the delivery of a decoy oligonucleotide (dODN) in human colon carcinoma SW 480 cells. This nanoparticle-dODN bioconjugate (γFe(2)O(3)@dODN) was designed to target the signal transducer and activator of transcription 3, STAT3, a key regulator of cell survival and proliferation. We exploited a simple precipitation-redispersion mechanism for the direct and one-step complexation of a labeled decoy oligonucleotide with iron oxide nanoparticles (NPs). The cell internalization of the decoy γFe(2)O(3)@dODN nanoparticles is demonstrated and suggests the potential for DNA delivery in biological applications. Despite the increasing use of NPs in biology and medicine, convenient methods to quantify them within cells are still lacking. In this work, taking advantage of the nonlinear magnetic behavior of our superparamagnetic NPs, we have developed a new method to quantify in situ their internalization by cells. FROM THE CLINICAL EDITOR: In this study, the authors demonstrate methods to quantify superparamagnetic nanocarriers within cells, taking advantage of the nonlinear magnetic behavior of the studied NPs.

Conformational transition of DNA bound to Hfq probed by infrared spectroscopy.

Hfq is a bacterial protein involved in RNA metabolism. Besides this, Hfq's role in DNA restructuring has also been suggested. Since this mechanism remains unclear, we examined the DNA conformation upon Hfq binding by combining vibrational spectroscopy and neutron scattering. Our analysis reveals that Hfq, which preferentially interacts with deoxyadenosine rich sequences, induces partial opening of dA-dT sequences accompanied by sugar repuckering of the dA strand and hence results in a heteronomous A/B duplex. Sugar repuckering is probably correlated with a global dehydration of the complex. By taking into account Hfq's preferential binding to A-tracts, which are commonly found in promoters, potential biological implications of Hfq binding to DNA are discussed.

Auto-assembly of E. coli DsrA small noncoding RNA: Molecular characteristics and functional consequences.

RNA molecules are important factors involved in different cellular processes and have a multitude of roles in the cell. These roles include serving as a temporary copy of genes used for protein synthesis or functions in translational machinery. Interestingly, RNA is so far the only biological molecule that serves both as a catalyst (like proteins) and as information storage (like DNA). However, in contrast to proteins well known to be able to self-associate in order to maintain the architecture of the cell, such RNA polymers are not prevalent in cells and are usually not favored by the flexibility of this molecule. In this work, we present evidence that such a polymer of a natural RNA, the DsrA RNA, exists in the bacterial cell. DsrA is a small noncoding RNA (87 nucleotides) of Escherichia coli that acts by base-pairing to mRNA in order to control the translation and the turnover of some mRNA, including rpoS mRNA, which encodes the sigma(s) RNA polymerase subunit involved in bacterial stress response. A putative model is proposed for the structure of this RNA polymer. Although the function of this polymerization is not known completely, we propose that the formation of such a structure could be involved in the regulation of DsrA ncRNA concentration in vivo or in a quality control mechanism used by the cell to eliminate misfolded RNAs.

Vibrational analysis of amino acids and short peptides in hydrated media. I. L-glycine and L-leucine.

Raman scattering and Fourier-transform infrared (FT-IR) attenuated transmission reflectance (ATR) spectra of two alpha-amino acids (alpha-AAs), i.e., glycine and leucine, were measured in H2O and D2O (at neutral pH and pD). This series of observed vibrational data gave us the opportunity to analyze vibrational features of both AAs in hydrated media by density functional theory (DFT) calculations at the B3LYP/6-31++G* level. Harmonic vibrational modes calculated after geometry optimization on the clusters containing each AA and 12 surrounding water molecules, which represent primary models for hydration scheme of amino acids, allowed us to assign the main observed peaks.

FTIR and UV spectroscopy studies of triplex formation between alpha-oligonucleotides with non-ionic phoshoramidate linkages and DNA targets.

The triplexes formed by pyrimidine alpha-oligodeoxynucleotides, 15mers alpha dT(15) or 12mers alpha dCT having dimethoxyethyl (PNHdiME), morpholino (PMOR) or propyl (PNHPr) non-ionic phosphoramidate linkages with DNA duplex targets have been investigated by UV and FTIR spectroscopy. Due to the decrease in the electrostatic repulsion between partner strands of identical lengths all modifications result in triplexes more stable than those formed with unmodified phosphodiester beta-oligodeoxynucleotides (beta-ODNs). Among the alpha-ODN third strands having C and T bases and non-ionic phosphoramidate linkages (alpha dCTPN) the most efficient modification is (PNHdiME). The enhanced third strand stability of the alpha dCTPN obtained as diastereoisomeric mixtures is attenuated by the steric hindrance of the PMOR linkages or by the hydrophobicity of the PNHPr linkages. All alpha dCTPN strands form triplexes even at neutral pH. In the most favorable case (PNHdiME), we show by FTIR spectroscopy that the triplex formed at pH 7 is held by Hoogsteen T*A.T triplets and in addition by an hydrogen bond between O6 of G and C of the third strand (Tm = 30 degrees C). The detection of protonated cytosines is correlated at pH 6 with a high stabilization of the triplex (Tm = 65 degrees C). While unfavorable steric effects are overcome with alpha anomers, the limitation of the pH dependence is not completely suppressed. Different triplexes are evidenced for non pH dependent phosphoramidate alpha-thymidilate strands (alpha dT(15)PN) interacting with a target duplex of identical length. At low ionic strength and DNA concentration we observe the binding to beta dA(15) either of alpha dT(15)PN as duplex strand and beta dT(15) as third strand, or of two hydrophobic alpha dT(15)PNHPr strands. An increase in the DNA and counterion concentration stabilizes the anionic target duplex and then the alpha dT(15)PN binds as Hoogsteen third strand.

FTIR and UV spectroscopy studies of triplex formation between pyrimidine methoxyethylphosphoramidates alpha-oligodeoxynucleotides and ds DNA targets.

The ability of non-ionic methoxyethylphosphoramidate (PNHME) alpha-oligodeoxynucleotides (ODNs), alpha dT(15) and alpha dCT dodecamer, to form triplexes with their double-stranded DNA targets was evaluated. Thermal stability of the formed complexes was studied by UV thermal denaturation and the data showed that these PNHME alpha-ODNs formed much more stable triplexes than phosphodiester (PO) beta-ODNs did (Delta Tm = + 20 degrees C for alpha dCT PNHME). In addition, FTIR spectroscopy was used to determine the base pairing and the strand orientations of the triplexes formed by alpha dT(15) PNHME compared to phosphodiester ODNs with beta or alpha anomeric configuration. While beta dT(15) PO failed to form a triplex with a long beta dA(n) x beta dT(n) duplex, the Tm of the Hoogsteen part of the triplex formed by alpha dT(15) PNHME reached 40 degrees C. Moreover alpha dT(15) PNHME displaced the beta dT(15) strand of a shorter beta dA(15) x beta dT(15) duplex. The alpha dCT PNHME and alpha dT(15) PNHME third strands were found antiparallel in contrast to alpha dT(15) PO which is parallel to the purine strand of their duplex target. The uniform preferential Hoogsteen pairing of the nucleotides alpha dT and alpha dC combining both replacements might contribute to the improve stability of the triplexes.