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Circular dichroism (CD) spectroscopy is a fast and simple technique providing important information about the conformation of nucleic acids, proteins, sugars, lipids, and their interactions between each other. This electronic absorption spectroscopy method is extremely sensitive to any change in molecular structure containing asymmetric molecules. While numerous reviews describe how to analyze deoxyribonucleic acid (DNA) structures using CD, analyses of ribonucleic acids (RNAs) are scarce. Nevertheless, RNAs are important molecules involved in a multitude of roles in the cell. In this chapter, we present applications of synchrotron radiation circular dichroism (SRCD) extending the spectral range down to 170 nm, improving structural analysis of RNA, including the analysis of helical parameters and alternative structures found in RNA. The effects of temperature to measure thermodynamic parameters and analyze ribonucleoprotein complexes will also be presented.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.Native electrospray ionization mass spectrometry (native ESI-MS) is a powerful tool to investigate non-covalent biomolecular interactions. It has been widely used to study protein complexes, but only few examples are described for the analysis of complexes involving RNA-RNA interactions. Here, we provide a detailed protocol for native ESI-MS analysis of RNA complexes. As an example, we present the analysis of the HIV-1 genomic RNA dimerization initiation site (DIS) extended duplex dimer bound to the aminoglycoside antibiotic lividomycin.RNA modification mapping by mass spectrometry (MS) is based on the use of specific ribonucleases (RNases) that generate short oligonucleotide digestion products which are further separated by nano-liquid chromatography and analyzed by MS and MS/MS. Recent developments in MS instrumentation allow the possibility to deeply explore posttranscriptional modifications. Notably, development of nano-liquid chromatography and nano-electrospray drastically increases the detection sensitivity and allows the identification and sequencing of RNA digested fragments separated and extracted from two-dimensional polyacrylamide gels, as long as the mapping and characterization of ribonucleotide modifications.We have previously described (Geffroy et al. Methods Mol Biol 166525-40, 2018) how to unfold (or fold) a single RNA molecule under force using a dual-beam optical trap setup. this website In this chapter, we complementarily describe how to analyze the corresponding data and how to interpret it in terms of RNA three-dimensional structure. As with all single-molecule methods, single RNA molecule force data often exhibit several discrete states where state-to-state transitions are blurred in a noisy signal. In order to cope with this limitation, we have implemented a novel strategy to analyze the data, which uses a hidden Markov modeling procedure. A representative example of such an analysis is presented.Surface plasmon resonance (SPR)-based instruments have become gold-standard tools for investigating molecular interactions involving macromolecules. The major advantage is that the measured signal is sensitive to changes in mass. Therefore, all kinds of complexes can be analyzed including those with compounds as small as cations. SPR is mainly used to determine the dissociation equilibrium constant and the binding rates of a reaction if slow enough. SPR is well suited for analysis molecular interactions with nucleic acids because these negatively charged macromolecules do not have a tendency to stick to the sensor chip surface as some proteins can do. To illustrate the use of SPR with RNA molecules, we describe methods that we used for monitoring the interaction between the protein Rop from E. coli and two RNA-RNA loop-loop complexes. One is derived from the natural target of Rop, RNAI-RNAII. The other one is an RNA-RNA complex formed between a shortened version of the TAR element of HIV-1 and a structured RNA, TAR* rationally designed to interact with TAR through loop-loop interactions. These methods can be easily adapted to other complexes involving RNA molecules and to other SPR instruments.Data from fluorescence-based methods that measure in vivo hybridization efficacy of unique RNA regions can be used to infer regulatory activity and to identify novel RNA RNA interactions. Here, we document the step-by-step analysis of fluorescence data collected using an in vivo regional RNA structural sensing system (iRS3) for the purpose of identifying potential functional sites that are likely to be involved in regulatory interactions. We also detail a step-by-step protocol that couples this in vivo accessibility data with computational mRNA target predictions to inform the selection of potentially true targets from long lists of thermodynamic predictions.Dynamic light scattering represents an accurate, robust, and reliable technique to analyze molecule size in solution and monitor their interactions in real time. Here, we describe how to analyze by DLS an RNA-protein interaction. In our frame, we studied complexes formed between RNA fragments derived from the genome of HIV-1 in association with the viral precursor Pr55Gag. These interactions are crucial for the specific selection of the viral genomic RNA (gRNA) from the bulk of the viral spliced and cellular RNAs. This chapter displays how DLS allows to characterize the interactions that regulate the early steps of viral assembly.