Protein-nucleic Acid Interactions Research Articles

Proteomic Analysis Reveals Diverse Classes of Arginine Methylproteins in Mitochondria of Trypanosomes

Arginine (arg) methylation is a widespread posttranslational modification of proteins that impacts numerous cellular processes such as chromatin remodeling, RNA processing, DNA repair, and cell signaling. Known arg methylproteins arise mostly from yeast and mammals, and are almost exclusively nuclear and cytoplasmic. Trypanosoma brucei is an early branching eukaryote whose genome encodes five putative protein arg methyltransferases, and thus likely contains a plethora of arg methylproteins. Additionally, trypanosomes and related organisms possess a unique mitochondrion that undergoes dramatic developmental regulation and uses novel RNA editing and mitochondrial DNA replication mechanisms. Here, we performed a global mass spectrometric analysis of the T. brucei mitochondrion to identify new arg methylproteins in this medically relevant parasite. Enabling factors of this work are use of a combination digestion with two orthogonal enzymes, an efficient offline two dimensional chromatography separation, and high-resolution mass spectrometry analysis with two complementary activations. This approach led to the comprehensive, sensitive and confident identification and localization of methylarg at a proteome level. We identified 167 arg methylproteins with wide-ranging functions including metabolism, transport, chaperoning, RNA processing, translation, and DNA replication. Our data suggest that arg methylproteins in trypanosome mitochondria possess both trypanosome-specific and evolutionarily conserved modifications, depending on the protein targeted. This study is the first comprehensive analysis of mitochondrial arg methylation in any organism, and represents a significant advance in our knowledge of the range of arg methylproteins and their sites of modification. Moreover, these studies establish T. brucei as a model organism for the study of posttranslational modifications.

Aurintricarboxylic Acid Ameliorates Experimental Autoimmune Encephalomyelitis by Blocking Chemokine-Mediated Pathogenic Cell Migration and Infiltration

Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by the immune-mediated demyelination and neurodegeneration of the CNS. Overactivation of CD4(+) T cells, especially the Th1 and Th17 subpopulations, is thought to be the direct cause of this disease. Aurintricarboxylic acid (ATA), an inhibitor of protein-nucleic acid interaction, has been reported to block with the JAK/STAT signaling pathway that is critical for Th cell differentiation. In this study, we discovered that ATA treatment significantly reduces the clinical score of EAE, but it does not directly inhibit the differentiation of Th1 and Th17 cells in vitro. ATA was found to block the chemotaxis and accumulation of dendritic cells in the spleen of EAE mice before the onset of the disease and to reduce the percentage of Th1 and Th17 cells in the spleen. Further study revealed that ATA also blocks the infiltration of pathogenic T cells into the CNS and blocks the onset of passive EAE. ATA was found to inhibit the functions of many chemokine receptors. By blocking chemokine-mediated migration of dendritic cells and pathogenic T cells, ATA alleviates the pathogenesis of EAE and might be used to treat autoimmune diseases, including multiple sclerosis.

Max Bergmann lecture protein epitope mimetics in the age of structural vaccinology.

This review highlights the growing importance of protein epitope mimetics in the discovery of new biologically active molecules and their potential applications in drug and vaccine research. The focus is on folded β-hairpin mimetics, which are designed to mimic β-hairpin motifs in biologically important peptides and proteins. An ever-growing number of protein crystal structures reveal how β-hairpin motifs often play key roles in protein–protein and protein–nucleic acid interactions. This review illustrates how using protein structures as a starting point for small-molecule mimetic design can provide novel ligands as protein–protein interaction inhibitors, as protease inhibitors, and as ligands for chemokine receptors and folded RNA targets, as well as novel antibiotics to combat the growing health threat posed by the emergence of antibiotic-resistant bacteria. The β-hairpin antibiotics are shown to target a β-barrel outer membrane protein (LptD) in Pseudomonas sp., which is essential for the biogenesis of the outer cell membrane. Another exciting prospect is that protein epitope mimetics will be of increasing importance in synthetic vaccine design, in the emerging field of structural vaccinology. Crystal structures of protective antibodies bound to their pathogen-derived epitopes provide an ideal starting point for the design of synthetic epitope mimetics. The mimetics can be delivered to the immune system in a highly immunogenic format on the surface of synthetic virus-like particles. The scientific challenges in molecular design remain great, but the potential significance of success in this area is even greater. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

The physico-chemical “anatomy” of the tautomerization through the DPT of the biologically important pairs of hypoxanthine with DNA bases: QM and QTAIM perspectives

The biologically important tautomerization of the Hyp·Cyt, Hyp·Thy and Hyp·Hyp base pairs to the Hyp·Cyt, Hyp·Thy and Hyp·Hyp base pairs, respectively, by the double proton transfer (DPT) was comprehensively studied in vacuo and in the continuum with a low dielectric constant (ε = 4) corresponding to hydrophobic interfaces of protein-nucleic acid interactions by combining theoretical investigations at the B3LYP/6-311++G(d,p) level of QM theory with QTAIM topological analysis. Based on the sweeps of the energetic, electron-topological, geometric and polar parameters, which describe the course of the tautomerization along the intrinsic reaction coordinate (IRC), it was proved that the tautomerization through the DPT is concerted and asynchronous process for the Hyp·Cyt and Hyp·Thy base pairs, while concerted and synchronous for the Hyp·Hyp homodimer. The continuum with ε = 4 does not affect qualitatively the course of the tautomerization reaction for all studied complexes. The nine key points along the IRC of the Hyp·Cyt↔Hyp·Cyt and Hyp·Thy↔Hyp·Thy tautomerizations and the six key points of the Hyp·Hyp↔Hyp·Hyp tautomerization have been identified and fully characterized. These key points could be considered as electron-topological "fingerprints" of concerted asynchronous (for Hyp·Cyt and Hyp·Thy) or synchronous (for Hyp·Hyp) tautomerization process via the DPT. It was found, that in the Hyp·Cyt, Hyp·Thy, Hyp·Hyp and Hyp·Hyp base pairs all H-bonds are significantly cooperative and mutually reinforce each other, while the C2H…O2 H-bond in the Hyp·Cyt base pair and the O6H…O4 H-bond in the Hyp·Thy base pair behave anti-cooperatively, i.e., they become weakened, while two others become strengthened.

Positional effects of click cyclization on β-hairpin structure, stability, and function

The use of the copper(I)-assisted azide-alkyne cycloaddition (CuAAC, or "click" reaction) as a method of β-hairpin stabilization was investigated at several different positions to determine the impact on hairpin structure and function, including hydrogen bonded sites, non-hydrogen bonded sites, and at the peptide termini. The role of the turn sequence in the peptide and the chain length of the azide were also investigated. It was determined that the CuAAC reaction was a suitable method for locking in β-hairpin structure in peptides possessing either the type I' turn, VNGO and the type II' turn, VpGO. Moreover, all cyclic variants exhibited improved thermal stability and resistance to proteolysis as compared to the non-cyclic peptides, regardless of the position in the strand. Additionally, the function of the CuAAC cyclized peptides was not altered as exhibited by similar binding affinities for ATP as the WKWK peptide. These studies provide a comprehensive investigation of CuAAC cyclization of β-hairpin peptides, which could further be applied to the inhibition of protein-protein and protein-nucleic acid interactions.

β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation)

Immunoprecipitation (IP) is a widely used method to isolate a specific protein from a mixed protein sample using an antibody that exclusively binds to that particular protein. This technique allows studying protein-protein and protein-nucleic acid interactions or to identify post-translational protein modifications. Many proteins, in particular cell surface receptors, localize to different compartments within cells where they elicit distinct functions by interacting with specific proteins. Integrins represent a major family of cell surface receptors consisting of non-covalently associated α and β subunits that mediate the interaction of cells with their environment. However, integrins do not only localize to the cell surface but are also present in other compartments including the endoplasmic reticulum and endosomes where they engage with a distinct set of interacting partners or show distinct post-translational modifications. Standard immunoprecipitation of β1 integrins from a cell lysate without prior fractionation isolates β1 integrins from all compartments. In contrast, selective immunoprecipitation of cell surface β1 integrin allows enriching for the pool of β1 integrin on the cell surface thereby minimizing contaminations with β1 integrins from other subcellular compartments. To achieve this, living cells are incubated with a β1 integrin-specific antibody on ice to label cell surface β1 integrins prior to cell lysis and precipitation., 免疫沉淀（IP）是一种广泛使用的方法，使用仅与该特定蛋白结合的抗体从混合蛋白样品中分离特定蛋白。这种技术允许研究蛋白质 - 蛋白质和蛋白质 - 核酸相互作用或识别翻译后蛋白质修饰。许多蛋白质，特别是细胞表面受体，定位于细胞内的不同区室，其中它们通过与特定蛋白质相互作用引起不同的功能。整联蛋白代表由非共价缔合的α和β亚基组成的细胞表面受体的主要家族，其介导细胞与其环境的相互作用。然而，整联蛋白不仅定位于细胞表面，而且存在于其它区室中，包括内质网和内体，其中它们与不同组的相互作用配偶体接合或显示不同的翻译后修饰。来自细胞裂解物的β1整联蛋白的标准免疫沉淀，没有预先分级分离来自所有区室的β1整联蛋白。相比之下，细胞表面β1整合素的选择性免疫沉淀允许富集β1整联蛋白在细胞表面上的库，从而使来自其他亚细胞区室的β1整联蛋白的污染最小化。为了实现这一点，活细胞与β1整合素特异性抗体在冰上孵育以在细胞裂解和沉淀之前标记细胞表面β1整联蛋白。

Attractor Transformation by Impulsive Control in Boolean Control Network

Boolean control networks have recently been attracting considerable interests as computational models for genetic regulatory networks. In this paper, we present an approach of impulsive control for attractor transitions in Boolean control networks based on the recent developed matrix semitensor product theory. The reachability of attractors is estimated, and the controller is also obtained. The general derivation proposed here is exemplified with a kind of gene model, which is the protein-nucleic acid interactions network, on numerical simulations.

Hierarchical Accessibility of Double-Helical RNA and DNA Processing Signals in Highly Dense Self-Assemblies

The ability to accurately detect biomolecules in single-cell amounts is an important goal of basic and translational research. The inherent capability of nucleic acids to self-assemble allows the spontaneous formation of highly dense, self-assembled monolayers (SAMs). These assemblages can provide the basis for development of nano-based devices with programmable, single-molecule detection capability. Double-stranded(ds) RNA is an important biomarker of viral infection and certain cancers. While dsRNA behavior in solution has been extensively characterized by diverse physicochemical approaches, the properties of highly dense assemblies of dsRNA are largely unknown, and may be qualitatively different from those in solution. High-density, 39 bp, chimeric dsRNA:dsDNA (ds-chimera) SAMs have been prepared on modified gold surfaces. using atomic force microscopy (AFM)-based nanomanipulation and analysis, and fluorescence microscopy, we demonstrate the preferential binding of ethidium to the ds-chimera SAM compared to the single-stranded form. As revealed by AFM detection of height change, the ds-chimera SAM is reactive towards the dsRNA-specific RNase III of Aquifex aeolicus (Aa-RNase III) and restriction endonuclease BamHI, each having a recognition site in the dsRNA and dsDNA segments, respectively. We also show that the reactivity of the BamHI cleavage site, which is proximal to the gold surface, can be controlled by SAM density, and that access to the BamHI site is dependent upon the prior action of RNase III at its site in the center of the ds-chimera. These results reveal novel properties of protein-nucleic acid interactions within a high-density array environment that are relevant to nanoscale detection methodologies.

Analysis of RNA-protein Interactions Using Electrophoretic Mobility Shift Assay (Gel Shift Assay)

RNA binding proteins (RBPs) play a crucial role in regulating gene expression at the post-transcriptional level at multiple steps including pre-mRNA splicing, polyadenylation, mRNA stability, mRNA localization and translation. RBPs regulate these processes primarily by binding to specific sequence elements in nascent or mature transcripts. There are several hundreds of RBPs in plants, but the targets of most of them are unknown. A variety of experimental methods have been developed to identify targets of an RBP. These include RNA immunoprecipitation (RIP), UV cross-linking and immunoprecipitation (CLIP) and many variations of CLIP (e.g. PAR-CLIP, iCLIP). These approaches depend on immunoprecipitation of RNAs bound to a specific RBP using an antibody to that RBP. Electrophoretic mobility shift assay (EMSA), also called gel shift assay, has been used to analyze protein-nucleic acid interactions. It is a simple and powerful method to analyze protein-RNA/DNA interactions. In RNA EMSA, RNA-protein complexes are visualized by comparing the migration of RNA in the presence of a protein. Generally, in RNA EMSA a specific RNA sequence is used to analyze its interaction with a protein. In vitro transcribed 32P labeled or chemically synthesized RNA with a fluorescent tag is incubated with or without the protein of interest and the reaction mixture is then run on native polyacrylamide gel electrophoresis. RNA-Protein complexes migrate slowly as compared to free RNA, which can be visualized using an imaging system. In addition to test binding of an RBP to RNA, EMSA is also used to map the region in RNA and/or protein that is involved in interaction. Furthermore, the binding affinity can also be quantified using EMSA.

74 Novel geometric approaches to uniquely characterize DNA-binding interfaces

Protein–nucleic-acid interactions play a critical role in all steps of the gene expression pathway. DNA-binding proteins interact with DNA via distinct regions on their surface that are characterized by an ensemble of chemical, physical, and geometrical properties. In previous studies, we have developed unique approaches to characterize DNA-binding proteins combining geometric and/or electrostatic features (Stawiski et al., 2003; Shazman et al., 2007; Shazman & Mandel-Gutfreund, 2010). Here, I will present a novel approach we have recently developed to characterize protein structures by the distribution of their overlapping local surface patches. In this approach, the protein surface is represented by a bag of overlapping surface patches, which are defined by a central surface residue and its nearest surface neighbors. Using a similar approach to FragBag, a method used to retrieve protein structure neighbors based on short contiguous backbone segments (Budowski-Tal et al., 2010), we characterize each protein by a ‘bag-of-surface patches’ – a vector representing the distribution of different patches which appear on the protein surface. The similarity between two proteins is finally measured by the distance between their corresponding vectors of surface patches. Our results, based on a large benchmark of protein domains, show that the method has a great advantage over other methods for detecting remote function relationships between proteins based solely on the protein surface. We further show the applicability of the method to uniquely identify DNA-binding interfaces accurately and efficiently.

Differential Targeting of Unpaired Bases within Duplex DNA by the Natural Compound Clerocidin: A Valuable Tool to Dissect DNA Secondary Structure

Non-canonical DNA structures have been postulated to mediate protein-nucleic acid interactions and to function as intermediates in the generation of frame-shift mutations when errors in DNA replication occur, which result in a variety of diseases and cancers. Compounds capable of binding to non-canonical DNA conformations may thus have significant diagnostic and therapeutic potential. Clerocidin is a natural diterpenoid which has been shown to selectively react with single-stranded bases without targeting the double helix. Here we performed a comprehensive analysis on several non-canonical DNA secondary structures, namely mismatches, nicks, bulges, hairpins, with sequence variations in both the single-stranded region and the double-stranded flanking segment. By analysis of clerocidin reactivity, we were able to identify the exposed reactive residues which provided information on both the secondary structure and the accessibility of the non-paired sites. Mismatches longer than 1 base were necessary to be reached by clerocidin reactive groups, while 1-base nicks were promptly targeted by clerocidin; in hairpins, clerocidin reactivity increased with the length of the hairpin loop, while, interestingly, reactivity towards bulges reached a maximum in 3-base-long bulges and declined in longer bulges. Electrophoretic mobility shift analysis demonstrated that bulges longer than 3 bases (i.e. 5- and 7-bases) folded or stacked on the duplex region therefore being less accessible by the compound. Clerocidin thus represents a new valuable diagnostic tool to dissect DNA secondary structures.

Describing sequence-ensemble relationships for intrinsically disordered proteins.

Intrinsically disordered proteins participate in important protein-protein and protein-nucleic acid interactions and control cellular phenotypes through their prominence as dynamic organizers of transcriptional, post-transcriptional and signalling networks. These proteins challenge the tenets of the structure-function paradigm and their functional mechanisms remain a mystery given that they fail to fold autonomously into specific structures. Solving this mystery requires a first principles understanding of the quantitative relationships between information encoded in the sequences of disordered proteins and the ensemble of conformations they sample. Advances in quantifying sequence-ensemble relationships have been facilitated through a four-way synergy between bioinformatics, biophysical experiments, computer simulations and polymer physics theories. In the present review we evaluate these advances and the resultant insights that allow us to develop a concise quantitative framework for describing the sequence-ensemble relationships of intrinsically disordered proteins.

NPIDB: nucleic acid—protein interaction database

The Nucleic acid—Protein Interaction DataBase (http://npidb.belozersky.msu.ru/) contains information derived from structures of DNA–protein and RNA–protein complexes extracted from the Protein Data Bank (3846 complexes in October 2012). It provides a web interface and a set of tools for extracting biologically meaningful characteristics of nucleoprotein complexes. The content of the database is updated weekly. The current version of the Nucleic acid—Protein Interaction DataBase is an upgrade of the version published in 2007. The improvements include a new web interface, new tools for calculation of intermolecular interactions, a classification of SCOP families that contains DNA–binding protein domains and data on conserved water molecules on the DNA–protein interface.

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

The general transcription factor (TF) IIB is required for RNA polymerase (Pol) II initiation and extends with its B-reader element into the Pol II active centre cleft. Low-resolution structures of the Pol II-TFIIB complex indicated how TFIIB functions in DNA recruitment, but they lacked nucleic acids and half of the B-reader, leaving other TFIIB functions enigmatic. Here we report crystal structures of the Pol II-TFIIB complex from the yeast Saccharomyces cerevisiae at 3.4 Å resolution and of an initially transcribing complex that additionally contains the DNA template and a 6-nucleotide RNA product. The structures reveal the entire B-reader and protein-nucleic acid interactions, and together with functional data lead to a more complete understanding of transcription initiation. TFIIB partially closes the polymerase cleft to position DNA and assist in its opening. The B-reader does not reach the active site but binds the DNA template strand upstream to assist in the recognition of the initiator sequence and in positioning the transcription start site. TFIIB rearranges active-site residues, induces binding of the catalytic metal ion B, and stimulates initial RNA synthesis allosterically. TFIIB then prevents the emerging DNA-RNA hybrid duplex from tilting, which would impair RNA synthesis. When the RNA grows beyond 6 nucleotides, it is separated from DNA and is directed to its exit tunnel by the B-reader loop. Once the RNA grows to 12-13 nucleotides, it clashes with TFIIB, triggering TFIIB displacement and elongation complex formation. Similar mechanisms may underlie all cellular transcription because all eukaryotic and archaeal RNA polymerases use TFIIB-like factors, and the bacterial initiation factor sigma has TFIIB-like topology and contains the loop region 3.2 that resembles the B-reader loop in location, charge and function. TFIIB and its counterparts may thus account for the two fundamental properties that distinguish RNA from DNA polymerases: primer-independent chain initiation and product separation from the template.

Prototropic tautomerism and basic molecular principles of hypoxanthine mutagenicity: an exhaustive quantum-chemical analysis

The molecular structures, relative stability order, and dipole moments of a complete family of 21 planar hypoxanthine (Hyp) prototropic molecular–zwitterionic tautomers including ylidic forms were computationally investigated at the MP2/6–311++G(2df,pd)//B3LYP/6–311++G(d,p) level of theory in vacuum and in three different surrounding environments: continuum with a low dielectric constant (ϵ = 4) corresponding to a hydrophobic interface of protein–nucleic acid interactions, dimethylsulfoxide (DMSO), and water. The keto-N1HN7H tautomer was established to be the global minimum in vacuum and in continuum with ϵ = 4, while Hyp molecule exists as a mixture of the keto-N1HN9H and keto-N1HN7H tautomers in approximately equal amounts in DMSO and in water at T = 298.15 K. We found out that neither intramolecular tautomerization by single proton transfer in the Hyp base, nor intermolecular tautomerization by double proton transfer in the most energetically favorable Hyp·Hyp homodimer (symmetry C 2h ), stabilized by two equivalent N1H…O6 H-bonds, induces the formation of the enol tautomer (marked with an asterisk) of Hyp with cis-oriented O6H hydroxyl group relative to neighboring N1C6 bond. We first discovered a new scenario of the keto–enol tautomerization of Hyp · Hyp homodimer (C 2h ) via zwitterionic near-orthogonal transition state (TS), stabilized by N1+H…N1− and O6+H…N1− H-bonds, to heterodimer Hyp∗ · Hyp (C s ), stabilized by O6H…O6 and N1H…N1 H-bonds. We first showed that Hyp∗ · Thy mispair (C s ), stabilized by O6H…O4, N3H…N1, and C2H…O2 H-bonds, mimicking Watson–Crick base pairing, converts to the wobble Hyp · Thy base pair (C s ), stabilized by N3H…O6 and N1H…O2 H-bonds, via high- and low-energy TSs and intermediate Hyp · Thy∗, stabilized by O4H…O6, N1H…N3, and C2H…O2 H-bonds. The most energetically favorable TS is the zwitterionic pair Hyp+ · Thy− (C s ), stabilized by O6+H…O4−, O6+H…N3−, N1+H…N3−, and N1+H…O2− H-bonds. The authors expressed and substantiated the hypothesis, that the keto tautomer of Hyp is a mutagenic compound, while enol tautomer Hyp∗ does not possess mutagenic properties. The lifetime of the nonmutagenic tautomer Hyp∗ exceeds by many orders the time needed to complete a round of DNA replication in the cell. For the first time purine–purine planar H-bonded mispairs containing Hyp in the anti-orientation with respect to the sugar moiety – Hyp · Ade syn , Hyp · Gua∗ syn , and Hyp · Gua syn , that closely resembles the geometry of the Watson–Crick base pairs, have been suggested as the source of transversions. An influence of the surrounding environment (ϵ = 4) on the stability of studied complexes and corresponding TSs was estimated by means of the conductor-like polarizable continuum model. Electron-topological, structural, vibrational, and energetic characterictics of all conventional and nonconventional H-bonds in the investigated structures are presented. Presented data are key to understanding elementary molecular mechanisms of mutagenic action of Hyp as a product of the adenine deamination in DNA.

Single-Stranded DNA Curtains for Real-Time Single-Molecule Visualization of Protein–Nucleic Acid Interactions

Single-molecule imaging of biological macromolecules has dramatically impacted our understanding of many types of biochemical reactions. To facilitate these studies, we have established new strategies for anchoring and organizing DNA molecules on the surfaces of microfluidic sample chambers that are otherwise coated with fluid lipid bilayers. This previous work was reliant upon the use of double-stranded DNA, precluding access to information on biological processes involving single-stranded nucleic acid substrates. Here, we present procedures for aligning and visualizing single-stranded DNA molecules along the leading edges of nanofabricated barriers to lipid diffusion, in both "single-tethered" and "double-tethered" experimental formats. This new single-molecule approach provides long-awaited access to critical biological reactions involving single-stranded DNA binding proteins.

Assembly of DNA Curtains Using Hydrogen Silsesquioxane As a Barrier to Lipid Diffusion

We have established a single-molecule imaging experimental platform called "DNA curtains" in which DNA molecules tethered to a lipid bilayer are organized into patterns at nanofabricated metallic barriers on the surface of a microfluidic sample chamber. This technology has wide applications for real-time single-molecule imaging of protein-nucleic acid interactions. Here, we demonstrate that DNA curtains can also be made from hydrogen silsesquioxane (HSQ). HSQ offers important advantages over metallic barriers because it can be lithographically patterned directly onto fused silica slides without any requirement for further processing steps, thereby offering the potential for rapid prototype development and/or scale up for manufacturing.

Proteomics in Drug Discovery

Carbohydrate Vaccines- A burgeoning field of GlycomicsGaurav M. Doshi, Pradnya Palekar Shanbhag, Gayatri V.Aggarwal, Mukesh D.Shahare, Elvis A. Martis

Synthetic riboswitches for external regulation of genes transferred by replication-deficient and oncolytic adenoviruses

Therapeutic gene transfer by replication-defective viral vectors or, for cancer treatment, by replication-competent oncolytic viruses shows high promise for treatment of major diseases. To ensure safety, timing or dosing in patients, external control of therapeutic gene expression is desirable or even required. In this study, we explored the potential of artificial aptazymes, ligand-dependent self-cleaving ribozymes, as an innovative tool for regulation of therapeutic gene expression. Importantly, aptazymes act on RNA intrinsically, independent of regulatory protein–nucleic acid interactions and stoichiometry, are non-immunogenic and of small size. These are key advantages compared with the widely used inducible promoters, which were also reported to lose regulation at high copy numbers, e.g. after replication of oncolytic viruses. We characterized aptazymes in therapeutic gene transfer utilizing adenovectors (AdVs), adeno-associated vectors (AAVs) and oncolytic adenoviruses (OAds), which are all in advanced clinical testing. Our results show similar aptazyme-mediated regulation of gene expression by plasmids, AdVs, AAVs and OAds. Insertion into the 5′-, 3′- or both untranslated regions of several transgenes resulted in ligand-responsive gene expression. Notably, aptazyme regulation was retained during OAd replication and spread. In conclusion, our study demonstrates the fidelity of aptazymes in viral vectors and oncolytic viruses and highlights the potency of riboswitches for medical applications.

Interaction of Uracil and Uridine with the Cosolvent and Denaturant Aqueous Urea at Molecular Level: Effect of Na+, K+ and Ca++ Ions

For in silico drug protein–nucleic acids interaction studies, the change in thermodynamic parameters plays an important role, specifically for deriving QSAR equations. The present study is a step towards finding new thermodynamic descriptors. The characteristic changes in physico-chemical properties of uracil and its corresponding nucleoside–uridine, components of RNA, with urea having –CO–NH– function typical of proteins in the presence of alkali metal ions (Na+, K+ and Ca++) have been studied using ultrasonic, volumetric and viscometric techniques. Density, viscosity and ultrasonic velocity of the solutions of base/nucleoside and urea (10−4 mol l−1) in presence of salt solution (in the range 0.1–0.5 mol l−1) have been measured. The thermodynamic parameters have been deduced from the experimental data. This study has highlighted the role of the thermodynamic parameters in ligand–nucleic acid interactions at the molecular level.