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. © 2020, Springer Science+Business Media, LLC, part of Springer Nature

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 assembly»”, 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.

International audienceWe 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

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

1 - ArticleRaman 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

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

<div><p>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.</p></div

Preparation and characterization of synthetic melanin and peptide vaccine formulations.

<p>(a) Schematic of the chemical process for obtaining water-soluble peptide-melanin complexes. Evolution over time of b) the UV-visible spectrum during gp100-melanin synthesis and c) the absorbance ratio at 350 and 280 nm (A350/A280) for melanin (black squares) and for gp100-melanin (red circles). (d): TEM image of gp100-melanin after 18 h of incubation time (inset: high magnification). (e) SDS-PAGE showing the migration of the unbound gp100 peptide within the resolving gel after different incubation times in the presence of oxidizing L-Dopa leading to gp100-melanin formation (following electrophoresis, the gels were stained with Coomassie Blue). (f) FTIR spectra in deuterated solution for melanin (black line) and gp100-melanin (red line); the amide II' band at 1455 cm-1 (blue hatching) is characteristic of peptides.</p

Distribution of melanin in draining lymph nodes.

<p>Macroscopic aspect of the draining inguinal lymph nodes (arrows) of BALB/c mice 2 days after injections with gp100-melanin (a) or saline (b). Fontana-Masson staining of a draining lymph node 2 days after injection with gp100-melanin, showing melanin-laden macrophages in the sinuses (c) and in the paracortical area (d).</p

T-cell response after subcutaneous immunizations in C57BL/6 mice.

<p>Mice were immunized with pOVA35 or pOVA35-melanin (pOVA35-Mel), on days 0 and 14 and sacrificed on day 21. Splenocytes were re-stimulated in vitro either with a) the MHC class II epitope (CD4) or b) the MHC class I-epitope (CD8) (non conjugated to melanin). The numbers of IFNg-SFCs (Spot forming cells) were measured. Each point represents an individual mouse (n = 8 mice/group with pooled data from 2 different experiments of 4 mice each). Bars = median. ** p < 0.01 (Mann–Whitney test).</p

Phenotype of SIINFELKL-specific T-cells in mice immunized on days 0 and 14 with [pOVA30 + CpG] or [pOVA30-melanin + CpG].

<p>(a) gating strategy and representative results (in one mouse) for T-Bet, CD62L and granzyme expression. (b) mean ± SD expression of T-bet, CD62L and granzyme within the CD8⁺dextramer⁺ population (n = 8 mice/group, with pooled data from two different experiments of 4 mice each). ** p<0,01; ***p<0,001.</p