Interaction Of Small Molecules Research Articles

Measuring Small Molecule Binding to Escherichia coli AcrB by Surface Plasmon Resonance.

Antimicrobial resistance (AMR) is rapidly becoming one of the great healthcare challenges. A common mechanism employed by pathogenic bacteria to avoid the action of certain antibiotics is to overexpress efflux pumps that can extrude these drugs from the cell rendering them ineffective. Small molecule inhibitors that target bacterial efflux pumps provide a route toward reversing AMR. Here, we describe the application of surface plasmon resonance (SPR) technology to characterize protein:small molecule interactions between the inner membrane protein AcrB subunit of the Escherichia coli AcrA-AcrB-TolC efflux pump and its substrates and novel inhibitors. The SPR assay provides quantitative data about the kinetics of binding that can help guide the development of new chemotherapies to combat AMR.

PROMIS: Global Analysis of PROtein-Metabolite Interactions.

Small molecules are not only intermediates of metabolism, but also play important roles in signaling and in controlling cellular metabolism, growth, and development. Although a few systematic studies have been conducted, the true extent of protein-small molecule interactions in biological systems remains unknown. PROtein-metabolite interactions using size separation (PROMIS) is a method for studying protein-small molecule interactions in a non-targeted, proteome- and metabolome-wide manner. This approach uses size-exclusion chromatography followed by proteomics and metabolomics liquid chromatography-mass spectrometry analysis of the collected fractions. Assuming that small molecules bound to proteins would co-fractionate together, we found numerous small molecules co-eluting with proteins, strongly suggesting the formation of stable complexes. Using PROMIS, we identified known small molecule-protein complexes, such as between enzymes and cofactors, and also found novel interactions. © 2019 The Authors. Basic Protocol 1: Preparation of native cell lysate from plant material Support Protocol: Bradford assay to determine protein concentration Basic Protocol 2: Separation of molecular complexes using size-exclusion chromatography Basic Protocol 3: Simultaneous extraction of proteins and metabolites using single-step extraction protocol Basic Protocol 4: Metabolomics analysis Basic Protocol 5: Proteomics analysis.

Interaction of Phosphate with Lithium Cobalt Oxide Nanoparticles: A Combined Spectroscopic and Calorimetric Study.

Adsorption of small ions such as phosphates to the surfaces of metal oxides can significantly alter the behavior of these materials, especially when present in the nanoscale form. Lithium cobalt oxide is a good model system for understanding small-molecule interactions with emerging nanomaterials because of its widespread use in lithium ion batteries and its known activity as a water oxidation catalyst. Here, we present a thermodynamic analysis of phosphate adsorption to LiCoO2 and corroborate the results with additional in situ techniques, including zeta potential measurements and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, at pH values relevant to potential environmental release scenarios. Flow microcalorimetry measurements of phosphate interaction with LiCoO2 at pH 7.4 show that there are two distinct exothermic processes taking place. Time-sequence in situ ATR-FTIR with two-dimensional correlation analysis reveals the spectroscopic signatures of these processes. We interpret the data as an interaction of phosphate with LiCoO2 that occurs through the release of two water molecules and is therefore, best described as a condensation process rather than a simple adsorption, consistent with prior studies, demonstrating that phosphate interaction with LiCoO2 is highly irreversible. Additional measurements for over longer times of 5 months show that phosphate adsorption terminates with one surface layer and that continued transformation over longer periods of time arises from H+/Li+ exchange and slow transformation to a cobalt hydroxide, with phosphate adsorbed to the surface only. To the best of our knowledge, this is the first time that flow microcalorimetry and two-dimensional correlation spectroscopy have been applied in tandem to clarify the specific chemical reactions that occur at the interface of solids and adsorbates.

Identification of Tractable Drug-like eIF4Al Inhibitors with Potent Anti-Tumor Activity

Clinical efficacy of targeted signaling inhibitors for hematologic malignancies is limited bynoutgrowth of subpopulations with alternative pathways independent of the drug target. The eIF4F complex responsible for translation initiation is a convergence point for cancer-promoting signaling pathways and its inhibition leads to decreased expression of key oncoproteins and apoptosis. Lymphomas and leukemias show particular dependence on constitutive eIF4F activation. Indeed, natural compounds targeting the eIF4F enzymatic component, eIF4A1, demonstrate activities in vitro and in vivo against lymphoma and leukemia model systems, among other tumor types. The natural compound silvestrol is a potent inhibitor of eIF4A1, results in cancer cell cytotoxicity, and has an established therapeutic window in vivo. Silvestrol shows potent antitumor activity against 924 pan-cancer tumor cell lines with 830/924 (90%) sensitive at IC50 <100nM with lymphoma and leukemia cell lines being particularly sensitive. Silvestrol and other natural compounds, however, lack core drug-like properties and synthetic tractability. To discover new, specific and tractable inhibitors of eIF4A1 that are more drug-like, we have constructed several molecular models that we used to virtually screen more than 20 million compounds. eIF4A1 is the founding member of the DEAD-box RNA helicases, which include its paralogs eIF4A2 (91% amino-acid identity with eIF4A1) and eIF4A3 (60% identity). All DEAD-box helicases contain two RecA-like domains separated by a flexible linker. The cleft between these domains is lined with helicase motifs that mediate nucleotide binding and hydrolysis. In an absence of RNA or nucleotide, eIF4A proteins adopt diffuse open conformations; binding of RNA and ATP triggers transition to a more stable closed state. Modeling small-molecule interactions in the nucleotide cleft of eIF4A1 therefore assesses ability of molecules to lock eIF4A1 in a conformation unable to cycle through ATPase and helicase activities. A new crystal structure of eIF4A1 has become available (2019) with a resolution of 2 angstroms. The protein is co-crystallized with ANP in the nucleotide binding site at the interface of the N and C-terminal domains and with known inhibitor, Rocaglamide, bound to the interface of the eIF4A1and a polypurine RNA. We used this high-resolution crystal structure to build models predicting interactions of small molecules in the interdomain nucleotide-binding cleft. We then performed all-atom explicit-water molecular dynamics (MD) simulations for 500-700 ns to study conformational dynamics and atomic interactions of ATP-bound and ATP-unbound states. Extended molecular dynamic simulations confirm the hypothesis that rocaglamide stabilizes the interaction between the helicase and a polypurine sequence on RNA, thus preventing further ATPase activity and RNA unwinding. Pooling these results, we constructed two homology models of human eIF4A1 with both open and closed conformations as structural templates. Over 50 compounds identified as hits in silicowere ordered and tested thus far in our biochemical and cell-based validation platform. Using our machine learning and virtual screening approach targeted to the ATP binding site of eIF4A1, we identified a promising piperazine-amide fragment scaffold (UM107; ~300 MW) with similar electronics to nucleotide triphosphates. UM107 caused cellular toxicities with an LD50 of 50 uM and was weakly active in the biochemical screen against eIF4A1 with an IC50 of 250 uM. We will increase molecular weight by adding more groups to maximize hydrogen bond interactions in the active site. These analogs will be synthesized and screened virtually building on the core using established medicinal chemistry optimization tools followed by biochemical and cellular validation. We therefore have developed an accurate and novel in silico models of eIF4A1 highly useful in assessing interactions of small-molecule ATPase inhibitors, with focus on the ATP-binding cleft. Disclosures No relevant conflicts of interest to declare.

Targeting RNA with Small Molecules: Identification of Selective, RNA-Binding Small Molecules Occupying Drug-Like Chemical Space

Although the potential value of RNA as a target for new small molecule therapeutics is becoming increasingly credible, the physicochemical properties required for small molecules to selectively bind to RNA remain relatively unexplored. To investigate the druggability of RNAs with small molecules, we have employed affinity mass spectrometry, using the Automated Ligand Identification System (ALIS), to screen 42 RNAs from a variety of RNA classes, each against an array of chemically diverse drug-like small molecules (~50,000 compounds) and functionally annotated tool compounds (~5100 compounds). The set of RNA-small molecule interactions that was generated was compared with that for protein-small molecule interactions, and naïve Bayesian models were constructed to determine the types of specific chemical properties that bias small molecules toward binding to RNA. This set of RNA-selective chemical features was then used to build an RNA-focused set of ~3800 small molecules that demonstrated increased propensity toward binding the RNA target set. In addition, the data provide an overview of the specific physicochemical properties that help to enable binding to potential RNA targets. This work has increased the understanding of the chemical properties that are involved in small molecule binding to RNA, and the methodology used here is generally applicable to RNA-focused drug discovery efforts.

Identification of novel Nrf2/Keap1 pathway mutations in pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is a malignancy of lymphoid progenitor cells, characterized by a wide range of biological and clinical heterogeneity. Oxidative stress is a common problem observed in carcinogenesis and it is involved in developing treatment resistance. Nuclear Factor Erythroid-2-Like 2 (Nrf2) transcription factor is the main regulator of antioxidant responses. The levels of reactive oxygen species (ROS) are tightly controlled and regulated by Nrf2 and its suppressor protein Kelch-like ECH-associated protein 1 (Keap1). Recently, many studies have shown that most of the genes in the Nrf2/Keap1/nuclear factor kappa-B (NF-κB)/phosphotyrosine-independent ligand for the Lck SH2 domain Of 62 KDa (p62) pathway show abnormally high mutational variations in cancer. However, variations in the Nrf2/Keap1/NF-κB1/p62 pathway in pediatric ALL have not been thoroughly investigated, yet. Thirty children, who were diagnosed with pediatirc ALL were included in the study. The Nrf2/Keap1/NF-κB1/p62 pathway variants were analyzed by DNA sequencing analysis. The PolyPhen-2 program was used for identifying pathogenic mutations. Our study examined the molecular dynamics (MD) perspectives of the effect of A159T and E121K mutations on protein stability for the first time in the literature by using the GROMACS45 software package utilizing the OPSLAA force field. Of the detected 17 nucleotide changes, 6 were novel. The study predicted the potential pathological effect of two mutations p. A159T and p.E121K in the Keap1 gene. The MD perspectives revealed that the E121K mutant’s observed structural behavior accounted for the key role of His-129 and E121K, where E121K exhibited much higher drift compared to His-129. For a future perspective, it would be meaningful to study the protein–small molecule interactions of the Keap1 protein to elaborate on the drug effects in patients carrying these mutations.

Targeting G-quadruplexes with Organic Dyes: Chelerythrine-DNA Binding Elucidated by Combining Molecular Modeling and Optical Spectroscopy.

The DNA-binding of the natural benzophenanthridine alkaloid chelerythrine (CHE) has been assessed by combining molecular modeling and optical absorption spectroscopy. Specifically, both double-helical (B-DNA) and G-quadruplex sequences—representative of different topologies and possessing biological relevance, such as telomeric or regulatory sequences—have been considered. An original multiscale protocol, making use of molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations, allowed us to compare the theoretical and experimental circular dichroism spectra of the different DNA topologies, readily providing atomic-level details of the CHE–DNA binding modes. The binding selectivity towards G-quadruplexes is confirmed by both experimental and theoretical determination of the binding free energies. Overall, our mixed computational and experimental approach is able to shed light on the interaction of small molecules with different DNA conformations. In particular, CHE may be seen as the building block of promising drug candidates specifically targeting G-quadruplexes for both antitumoral and antiviral purposes.

Spatiochemically Profiling Microbial Interactions with Membrane Scaffolded Desorption Electrospray Ionization-Ion Mobility-Imaging Mass Spectrometry and Unsupervised Segmentation.

Imaging the inventory of microbial small molecule interactions provides important insights into microbial chemical ecology and human medicine. Herein we demonstrate a new method for enhanced detection and analysis of metabolites present in interspecies interactions of microorganisms on surfaces. We demonstrate that desorption electrospray ionization-imaging mass spectrometry (DESI-IMS) using microporous membrane scaffolds (MMS) enables enhanced spatiochemical analyses of interacting microbes among tested sample preparation techniques. Membrane scaffolded DESI-IMS has inherent advantages compared to matrix-assisted laser desorption ionization (MALDI) and other IMS methods through direct IMS analyses of microbial chemistry in situ. This rapid imaging method yields sensitive MS analyses with unique m/z measurements when compared to liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) via unmediated sampling by MMS DESI-IMS. Unsupervised segmentation imaging analysis of acquired DESI-IMS data reveals distinct chemical regions corresponding to intermicrobial phenomenon such as predation and communication. We validate the method by linking Myxovirescin A and DKxanthene-560 to their known biological roles of predation and phase variation, respectively. In addition to providing the first topographic locations of known natural products, we prioritize 54 unknown features using segmentation within the region of predation. Thus, DESI-IMS and unsupervised segmentation spatially annotates the known biology of myxobacteria and provides functional exploration of newly uncharacterized small molecules.

Using Small-Molecule Probes to Investigate Aggregation of Sunset Yellow FCF: What are the Concentration Limits?

The assembly of small molecules into larger structures, often driven by noncovalent interactions such as hydrogen bonding, aromatic stacking interactions, and burial of hydrophobic surface, is of widespread interest. The interaction of small molecules with aggregates also has a large range of applications from fluorescence aggregation assays to gas storage in framework materials. Here, we utilize nuclear magnetic resonance spectroscopy to investigate the interaction of a small-molecule probe on the assembly state of sunset yellow across a wide range of relative concentrations. Information from both macroscopic (diffusion) and microscopic (chemical shifts) measurements allows the interaction to be studied and the binding mode to be interrogated. Using fluorophenol as the small-molecule probe, we show that the aggregation behavior of sunset yellow is broadly unaffected by the relative amount of fluorophenol added.

Protein Interactions with Nanoparticle Surfaces: Highlighting Solution NMR Techniques.

In the last decade, nanoparticles (NPs) have become a key tool in medicine and biotechnology as drug delivery systems, biosensors and diagnostic devices. The composition and surface chemistry of NPs vary based on the materials used: typically organic polymers, inorganic materials, or lipids. Nanoparticle classes can be further divided into sub-categories depending on the surface modification and functionalization. These surface properties matter when NPs are introduced into a physiological environment, as they will influence how nucleic acids, lipids, and proteins will interact with the NP surface. While small-molecule interactions are easily probed using NMR spectroscopy, studying protein-NP interactions using NMR introduces several challenges. For example, globular proteins may have a perturbed conformation when attached to a foreign surface, and the size of NP-protein conjugates can lead to excessive line broadening. Many of these challenges have been addressed, and NMR spectroscopy is becoming a mature technique for in situ analysis of NP binding behavior. It is therefore not surprising that NMR has been applied to NP systems and has been used to study biomolecules on NP surfaces. Important considerations include corona composition, protein behavior, and ligand architecture. These features are difficult to resolve using classical surface and material characterization strategies, and NMR provides a complementary avenue of characterization. In this review, we examine how solution NMR can be combined with other analytical techniques to investigate protein behavior on NP surfaces.

Development of viscometric methods for studying the interaction of porphyrins with DNA

The present paper is an overview of studying of DNA-porphyrin interactions using viscometry in combination with the spectroscopic methods. It was shown, that when porphyrins interact with DNA as an outside binder, the interaction mode and intensity does not depend on metal center, peripheral substituent’s and their positions on pyridylic ring. In case of planar porphyrins, the binding type is mainly determined by type of peripheral substituent’s and their position on the pyridylic ring. Currently, viscometry is widely used to study the interaction of porphyrins with DNA as an adjunct to other methods. Due to high accuracy and maximum sensitivity to changes in the size and shape of macromolecules, it is recommended to use viscometry as the cogent method for studying the interaction of small molecules with DNA, especially if intercalation is expected using other methods if necessary to confirm the results obtained.AbbreviationsDNAdeoxyribonucleic acidH2TAllPyP4meso-tetra-(4N-allylpyridyl) porphyrinH2TAllPyP3meso-tetra-(3N-allylpyridyl) porphyrinH2THOEtPyP4meso-tetra-(4N-hydroxyethylpyridyl) porphyrinH2THOEtPyP3meso-tetra-(3N-hydroxyethylpyridyl) porphyrinUV/VIS spectrophotometryultraviolet-visible spectrophotometryCD spectroscopycircular dichroism spectroscopyCommunicated by Ramaswamy H. Sarma

Range-separated double-hybrid density-functional theory with coupled-cluster and random-phase approximations.

We construct range-separated double-hybrid (RSDH) schemes which combine coupled-cluster or random-phase approximations (RPAs) with a density functional based on a two-parameter Coulomb-attenuating-method-like decomposition of the electron-electron interaction. We find that the addition of a fraction of short-range electron-electron interaction in the wave-function part of the calculation is globally beneficial for the RSDH scheme involving a variant of the RPA with exchange terms. Even though the latter scheme is globally as accurate as the corresponding scheme employing only second-order Møller-Plesset perturbation theory for atomization energies, reaction barrier heights, and weak intermolecular interactions of small molecules, it is more accurate for the more complicated case of the benzene dimer in the stacked configuration. The present RSDH scheme employing a RPA thus represents a new member in the family of double hybrids with minimal empiricism which could be useful for general chemical applications.

Electroorganic Synthesis and the Construction of Addressable Molecular Surfaces.

In this short review, an electroorganic synthesis approach to the construction of addressable, complex molecular surfaces is described along with the parameters that guided the development of that synthetic approach. The result of the work is a synthetic toolbox that will allow microelectrode arrays to be used for the "real-time" monitoring of small molecule interactions with biological targets.

Spectroscopic studies of interactions of 2-(2-Oxo-2-Phenylethyl)-1, 2-benzisothiazol-3(2H)-one-1, 1-dioxide with human DNA

The interaction of small molecules with DNA has become the most prominent area of research for the discovery of new drugs that may be helpful in curing of the diseases. The present research work was designed to explore the interactions of 2-(2-Oxo-2-Phenylethyl)-1,2-benzisothiazol-3(2H)-one 1,1-dioxide (BTZDO) with DNA by spectroscopic methods in physiological buffers of pH 4.7 and 7.4. Spectral results showed the hypochromic and bathochromic shifts after addition of DNA. The binding constant (Kf) of 5.18 × 102 and 8.62 × 102 Lmol−1 were determined at pH 4.7 (stomach pH) and 7.4 (blood pH) respectively. Strong values of binding constants reflected strong complex between BTZDO and DNA. The observed values of binding constant suggested that the intake of BTZDO inside human body via blood is more effective as compared to the oral intake. The interpretation of physical mode to intake the newly synthesized BTZDO drug into human body is the novelty of this research work. An intercalation binding of the BTZDO with DNA was supported by the results obtained from DNA melting studies. The Gibbs free energy calculated at pH 4.7 was −15.22 kJmol-1 and at pH 7.4 was −16.46 kJmol-1. The negative values of free energies are the prerequisite that the binding process to be spontaneous. Thermodynamic parameters (enthalpy and entropy) were also calculated. The enthalpy calculated for this system was −29.23 kJmol−1K−1 at pH 4.7 and −52.56 kJmol−1K−1 at pH 7.4. The entropy calculated for present system at pH 4.7 was −48.22 Jmol−1K−1 and at 7.4 the value was −122.89 Jmol−1K−1. Negative values of entropy predicted that a compact complex was formed between BTZDO and DNA. The focal point of this investigation was the observation of the effect of temperature on binding between the BTZDO and DNA, nature of complex formed and especially, the preferred mode adopted for the intake of medicine.

The Outliers: Metal-Mediated Radical Reagents for Biological Substrate Degradation.

The predictable and controllable interaction of small organic or peptidic molecules with biological substrates is the primary reason most pharmaceuticals are narrowly decorated carbon frameworks. The inhibition or activation binding models are measurable and without side reactions that can cause pathological angst. Yet many diseases, especially those involving rapid proliferation of cells (i.e., cancer) or aggregation of peptides (e.g., heart disease, Alzheimer's disease) have not yet been cured by inhibition therapeutics. Additionally, interventional medicine is often required to alleviate such maladies by physical removal first, followed by molecular-level therapy as a second stage. Thus, there appears to be a niche for more aggressive therapeutics that may employ harsher chemical processes to realize clinical efficacy, albeit without causing catastrophic side effects. Molecules that may be considered for this challenge are not typically biomimetic, nor do they fit the traditional pharmaceutical paradigm. They may have unusual modes of action or undesired reactivity that can be lethal if not controlled. These are the outliers; potential pharmacophores that biology does not know how to manage or adapt to. This is why they may be an intriguing class of agents that needs continuous development. In this Account, we connect the under-developed enediyne family of compounds and our metalloenediyne derivatives to existing radical-based therapeutics such as bleomycin and doxorubicin to illustrate that controlled diradical reactivity, although an outlier mechanism, has a place in the therapeutic portfolio. This is self-evident in that of the 11 natural product enediynes known, 2 have clinical impact, a strong ratio. We expand on the chemical diversity of potential enediyne constructs and focus on the accessible trigger mechanisms to activate diradical formation as a method to control toxicity. Moreover, we further illustrate how electromagnetic fields can be employed to activate both molecular and larger nanomaterial constructs that carry highly concentrated payloads of reactive reagent. Finally, we describe how controlled diradical reactivity can reach beyond traditional therapeutic targets such as DNA, to peptide aggregates found in blood clots, neural fibrils, and membrane scaffolds. It is our belief that cleverly constructed frameworks with well-designed and controlled activation/reaction schemes can lead to novel therapeutics that can challenge evolving viral and bacterial invaders. From this evangelical perspective, our hope is that the conceptual framework, if not the specific designs in this Account, stimulate the readership to develop out-of-the-box therapeutic designs that may combat resistant disease targets.

Structure-based design of potent linear peptide inhibitors of the YAP-TEAD protein-protein interaction derived from the YAP omega-loop sequence.

The YAP-TEAD protein-protein interaction is a potential therapeutic target to treat cancers in which the Hippo signaling pathway is deregulated. However, the extremely large surface of interaction between the two proteins presents a formidable challenge for a small molecule interaction disrupter approach. We have accomplished progress towards showing the feasibility of this approach by the identification of a 15-mer peptide able to potently (nanomolar range) disrupt the YAP-TEAD interaction by targeting only one of the two important sites of interaction. This peptide, incorporating non-natural amino acids selected by structure-based design, is derived from the Ω-loop sequence 85-99 of YAP.

Molecular Scale Optimum Hydrophobicity To Establish an Enhanced Probe-Protein Interaction: Near-Infrared Imaging of Albumin Biosynthesis Modulation.

Albumin is the most abundant serum protein and shows variation in its synthesis rate in different physiological and pathophysiological conditions. Thus, there might be an association expected between serum albumin concentration and body health. A library of NIR probes engineered with the optimum hydrophobicity has been developed and characterized using spectroscopy techniques and was employed to understand the variation of hepatic albumin synthesis rates on physiological and pathophysiological states. Given the importance of hydrophobicity in rendering an effective interaction of small molecules with biomolecules, strategic structure interaction relationship studies led us toward the development of a potent emissive molecular probe through chemical library development. By exploration of these newly developed molecular probes, our study elegantly showed how a pathophysiological condition like the hyperinsulinemic state significantly downregulates albumin biosynthesis in HepG2 cells using fluorescence microscopy as a tool. An excellent correlation between the albumin transcript level and fluorescence intensity inside the cells has been observed. The key role of hydrophobicity resulting in an effective interaction of the probes with albumin, thus leading to strong optical signals, has been experimentally demonstrated in this report. Also, a siRNA interference technique has been utilized to establish the excellent selectivity of the developed probes with excitation as well as emission in the NIR region. We therefore have established through our experimental findings that suitable cell permeable emissive molecular markers with a high degree of albumin specificity can be used as a good optical tool for studying the effect of hyperinsulinemia on albumin biosynthesis modulation.

Interaction of small molecules with polynucleotide repeatsand frameshift site RNA.

This mini-review describes the interaction between small molecules and RNA, in addition to its application either in treating RNA-associated diseases or detecting target molecules. In the case of RNA-associated diseasetreatment, the designed small molecules interact with RNA sites, forming adducts and providing successful therapeutic strategies over oligonucleotides. On the other hand, synthetically designed RNA moieties (aptamers) interact with target molecules like toxins, drugs, hormones; these interactions are useful in the detection, quantification or separation of these target moieties.

NMR characterization of RNA small molecule interactions.

Exciting discoveries of naturally occurring ligand-sensing and disease-linked noncoding RNAs have promoted significant interests in understanding RNA-small molecule interactions. NMR spectroscopy is a powerful tool for characterizing intermolecular interactions. In this review, we describe protocols and approaches for applying NMR spectroscopy to investigate interactions between RNA and small molecules. We review protocols for RNA sample preparation, methods for identifying RNA-binding small molecules, approaches for mapping RNA-small molecule interactions, determining complex structures, and characterizing binding kinetics. We hope this review will provide a guideline to streamline NMR applications in studying RNA-small molecule interactions, facilitating both basic mechanistic understandings of RNA functions and translational efforts in developing RNA-targeted therapeutics.

Biosensor-surface plasmon resonance: A strategy to help establish a new generation RNA-specific small molecules.

Biosensor surface plasmon resonance (SPR) is a highly sensitive technique and is most commonly used to decipher the interactions of biological systems including proteins and nucleic acids. Throughout the years, there have been significant efforts to develop SPR assays for studying protein-protein interactions, protein-DNA interactions, as well as small molecules to target DNAs that are of therapeutic interest. With the explosion of discovery of new RNA structures and functions, it is time to review the applications of SPR to RNA interaction studies, which have actually extended over a long time period. The primary advantage of SPR is its ability to measure affinities and kinetics in real time, along with being a label-free technique and utilizing relatively small quantities of materials. Recently, developments that use SPR to analyze the interactions of different RNA sequences with proteins and small molecules demonstrate the versatility of SPR as a powerful method in the analysis of the structure-function relationships, not only for biological macromolecules but also for potential drug candidates. This chapter will guide the reader through some background material followed by an extensive assay development to dissect the interactions of small molecules and RNA sequences using SPR as the critical method. The protocol includes (i) fundamental concepts of SPR, (ii) experimental design and execution, (iii) the immobilization of RNA using the streptavidin-biotin capturing method, and (iv) affinities and kinetics analyses of the interactions using specific example samples. The chapter also contains useful notes to address situations that might arise during the process. This assay demonstrates SPR as a valuable quantitative method used in the search for potential therapeutic agents that selectively target RNA.