Effect of tea catechins on tRNA structure and dynamics.

AbbreviationsCcatechinECGepicatechin gallateEGCGEpigallocatechin gallateAAdenineCcytosineGGuanineUuracilFTIRFourier transform infraredCommunicated by Ramaswamy H. Sarma.

G-Quadruplex Structure Improves the Immunostimulatory Effects of CpG Oligonucleotides.

Single-strand oligodeoxynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine (CpG) are recognized by the toll-like receptor 9, a component of the innate immunity. Therefore, they could act as immunotherapeutic agents. Chemically modified CpG ODNs containing a phosphorothioate backbone instead of phosphodiester (PD) were developed as immunotherapeutic agents resistant to nuclease degradation. However, they cause adverse side effects, and so there is a necessity to generate novel CpG ODNs. In the present study, we designed a nuclease-resistant nonmodified CpG ODN that forms G-quadruplex structures. G-quadruplex formation in CpG ODNs increased nuclease resistance and cellular uptake. The CpG ODNs designed in this study induced interleukin-6 production in a human B lymphocyte cell line and human peripheral blood mononuclear cells. These results indicate that G-quadruplex formation can be used to increase the immunostimulatory activity of CpG ODNs having a natural PD backbone.

Antimicrobial potential of myristic acid against Listeria monocytogenes in milk.

Listeria monocytogenes (L. monocytogenes), an important food-borne pathogenic microorganism, has resistance immune function to many commonly used drugs. Myristic acid is a traditional Chinese herbal medicine, but it has been rarely used as a food additive, limiting the development of natural food preservatives. In this study, the antibacterial activity and mechanism of myristic acid against L. monocytogenes were studied. The minimum inhibitory concentration (MIC) of myristic acid against 13 L. monocytogenes strains ranged from 64 to 256 µg ml-1. The time-kill assay demonstrated that when myristic acid was added to dairy products, flow cytometry confirmed that myristic acid influenced cell death and inhibited the growth of L. monocytogenes. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and NPN uptake studies illustrated that myristic acid changed the bacterial morphology and membrane structure of L. monocytogenes, which led to rapid cell death. Myristic acid could bind to DNA and lead to changes in DNA conformation and structure, as identified by fluorescence spectroscopy. Our studies provide additional evidence to support myristic acid being used as a natural antibacterial agent and also further fundamental understanding of the modes of antibacterial action.

Targeting RNA-protein interactions within the human immunodeficiency virus type 1 lifecycle.

RNA-protein interactions are vital throughout the HIV-1 life cycle for the successful production of infectious virus particles. One such essential RNA-protein interaction occurs between the full-length genomic viral RNA and the major structural protein of the virus. The initial interaction is between the Gag polyprotein and the viral RNA packaging signal (psi or Ψ), a highly conserved RNA structural element within the 5'-UTR of the HIV-1 genome, which has gained attention as a potential therapeutic target. Here, we report the application of a target-based assay to identify small molecules, which modulate the interaction between Gag and Ψ. We then demonstrate that one such molecule exhibits potent inhibitory activity in a viral replication assay. The mode of binding of the lead molecules to the RNA target was characterized by ¹H NMR spectroscopy.

Conformation-dependent DNA damage induced by gold nanoparticles.

The sensitivity of two conformations of plasmid DNA, the A and B forms, to strand break formation induced by gold nanoparticles (GNPs) is investigated by varying the GNP to DNA ratio in solution and the degree of DNA hydration. Decreasing DNA hydration via lyophilisation or by replacement of water with ethanol in solution modifies its conformation from the B to the A form. The yields of single strand breaks (SSB) are found to be highly dependent on the amount of DNA in the A configuration. The damage also increases with the amount of GNPs bound to DNA. At a ratio of two GNPs for one plasmid in an 80%-ethanol, 20%-water solution, 50% of the initial supercoiled population is converted to SSB. Thus, close contact with GNPs causes extensive damage to DNA in the A form. Since during transcription the DNA-RNA duplexes adopt an A form, GNPs could be genotoxic. Our results suggest that GNPs may have potential as chemotherapeutic agents if conjugated to nuclear targeting ligands. Considering their additional radiotherapeutic properties, targeted GNPs could also become highly effective in the treatment of cancer with concomitant chemoradiation therapy.

Interactions of CT-DNA with Hypocrellin A and its Al(3+)-Hypocrellin A complex.

In this study, the chelation of Hypocrellin A (HA) with Al(3+) in water solution has been synthesized, and the interactions of HA and Al(3+)-HA complex with calf thymus DNA are in detail compared by UV-vis and fluorescence spectroscopic techniques, circular dichroism spectroscopy and viscosity measurement. The experiment results suggest that HA and Al(3+)-HA complex both could bind to CT DNA by intercalation mode, and double helix of DNA was damaged. Moreover, Al(3+)-HA complex not only displays higher absorption at therapeutic window but also displays stronger binding affinity to CT DNA than HA.

Hidden complexity in the isomerization dynamics of Holliday junctions.

A plausible consequence of the rugged folding energy landscapes inherent to biomolecules is that there may be more than one functionally competent folded state. Indeed, molecule-to-molecule variations in the folding dynamics of enzymes and ribozymes have recently been identified in single-molecule experiments, but without systematic quantification or an understanding of their structural origin. Here, using concepts from glass physics and complementary clustering analysis, we provide a quantitative method to analyse single-molecule fluorescence resonance energy transfer (smFRET) data, thereby probing the isomerization dynamics of Holliday junctions, which display such heterogeneous dynamics over a long observation time (T(obs) ≈ 40 s). We show that the ergodicity of Holliday junction dynamics is effectively broken and that their conformational space is partitioned into a folding network of kinetically disconnected clusters. Theory suggests that the persistent heterogeneity of Holliday junction dynamics is a consequence of internal multiloops with varying sizes and flexibilities frozen by Mg(2+) ions. An annealing experiment using Mg(2+) pulses lends support to this idea by explicitly showing that interconversions between trajectories with different patterns can be induced.

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.

Identification of RNA pseudoknot-binding ligand that inhibits the -1 ribosomal frameshifting of SARS-coronavirus by structure-based virtual screening.

Programmed -1 ribosomal frameshifting (-1 RF) is an essential regulating mechanism of translation used by SARS-CoV (severe acute respiratory syndrome coronavirus) to synthesize the key replicative proteins encoded by two overlapping open reading frames. The integrity of RNA pseudoknot stability and structure in the -1 RF site is important for efficient -1 RF. Thus, small molecules interacting with high affinity and selectivity with the RNA pseudoknot in the -1 RF site of SARS-CoV (SARS-pseudoknot) would disrupt -1 RF and be fatal to viral infectivity and production. To discover ligands for the SARS-pseudoknot by virtual screening, we constructed a 3D structural model of the SARS-pseudoknot and conducted a computational screening of the chemical database. After virtual screening of about 80,000 compounds against the SARS-pseudoknot structure, high-ranked compounds were selected and their activities were examined by in vitro and cell-based -1 RF assay. We successfully identified a novel ligand 43 that dramatically inhibits the -1 RF of SARS-CoV. This antiframeshift agent is an interesting lead for the design of novel antiviral agents against SARS-CoV.

Interaction of ß-carboline alkaloids with RNA.

ß-Carboline alkaloids are present in medicinal plants such as Peganum harmala L., which have been used as folk medicine in anticancer therapy. Recently, they have drawn attention because of their antitumor activities. Despite considerable interest and investigations on alkaloid-DNA complexes, reports on alkaloid-RNA interaction are very limited. This study is the first attempt to investigate the binding of ß-carboline alkaloids (harmine, harmane, harmaline, harmalol, and tryptoline) with yeast RNA. The effect of alkaloid complexation on RNA aggregation and condensation was investigated in aqueous solution at physiological conditions, using constant RNA concentration (6.25 mM) and various alkaloid:polynucleotide (phosphate) ratios of 1:240, 1:160, 1:80, 1:40, 1:20, 1:10, 1:5, 1:2, and 1:1. Fourier transform infrared and UV-visible spectroscopic methods were used to determine the ligand-binding modes, the binding constants, and the stability of alkaloid-RNA complexes in aqueous solution. Spectroscopic evidence showed major binding of alkaloids to RNA with overall binding constants of K(harmine)-RNA = 2.95 × 107 M⁻¹, K(harmane)-RNA = 5.62 × 105 M⁻¹, K(harmaline)-RNA = 7.47 × 105 M⁻¹, K(harmalol)-RNA = 4.32 × 105 M⁻¹, and K(tryptoline)-RNA = 3.21 × 105 M⁻¹. The affinity of alkaloids-RNA binding is in the order of harmine > harmaline > harmane > harmalol > tryptoline. No biopolymer secondary structural changes were observed upon alkaloid interaction and RNA remains in the A-family structure in these complexes.

Curcumin protects DNA damage in a chronically arsenic-exposed population of West Bengal.

Groundwater arsenic contamination has been a health hazard for West Bengal, India. Oxidative stress to DNA is recognized as an underlying mechanism of arsenic carcinogenicity. A phytochemical, curcumin, from turmeric appears to be potent antioxidant and antimutagenic agent. DNA damage prevention with curcumin could be an effective strategy to combat arsenic toxicity. This field trial in Chakdah block of West Bengal evaluated the role of curcumin against the genotoxic effects of arsenic. DNA damage in human lymphocytes was assessed by comet assay and fluorescence-activated DNA unwinding assay. Curcumin was analyzed in blood by high performance liquid chromatography (HPLC). Arsenic induced oxidative stress and elucidation of the antagonistic role of curcumin was done by observation on reactive oxygen species (ROS) generation, lipid peroxidation and protein carbonyl. Antioxidant enzymes like catalase, superoxide dismutase, glutathione reductase, glutathioneS-transferase, glutathione peroxidase and non-enzymatic glutathione were also analyzed. The blood samples of the endemic regions showed severe DNA damage with increased levels of ROS and lipid peroxidation. The antioxidants were found with depleted activity. Three months curcumin intervention reduced the DNA damage, retarded ROS generation and lipid peroxidation and raised the level of antioxidant activity. Thus curcumin may have some protective role against the DNA damage caused by arsenic.

Co-exposure to arsenic and fluoride on oxidative stress, glutathione linked enzymes, biogenic amines and DNA damage in mouse brain.

We studied the effects of combined exposure to arsenic and fluoride on (i) brain biogenic amines, oxidative stress and its correlation with glutathione and linked enzymes; (ii) alterations in the structural integrity of DNA; and (iii) brain and blood arsenic and fluoride levels. Efficacy of alpha-tocopherol in reducing these changes was also determined. Male mice were exposed to sodium meta arsenite (50 ppm) and sodium fluoride (50 ppm) individually and in combination for ten weeks. Animals were given vitamin E supplementation (5 mg/kg, i.m., alternate days) throughout the experiment. Exposure to arsenic and fluoride significantly decreased the levels of brain biogenic amines. However; acetyl cholinesterase (AChE) and monoamine oxidase (MAO) activities showed an increase on fluoride exposure. There was also an increase in reactive oxygen species, thiobarbituric acid reactive species level, glutathione S-transferase and glutathione peroxidase activities and decreased superoxide dismutase activity, GSH:GSSG ratio, glucose 6-phosphate dehydrogenase activity. Combined exposure to these toxicants produced more pronounced effects on AChE, MAO, SOD and catalase activities. Infrared spectra showed less toxicity during combined exposure as the characteristic peaks of cytosine and alpha-helical structure of DNA were observed in normal and arsenic plus fluoride-exposed animals. Vitamin E reduced brain fluoride level and tissue oxidative stress but had no effect on arsenic. Combined exposure to arsenic and fluoride does not necessarily lead to more pronounced toxicity and interestingly exhibit some antagonistic effects. Vitamin E supplementation may be of added value in reverting some of the toxic effects.

Tertiary structure of an RNA pseudoknot is stabilized by "diffuse" Mg2+ ions.

The aim of this study is to obtain a comprehensive experimental and theoretical description of the contributions of Mg2+ ions to the free energy of folding a pseudoknot RNA tertiary structure. A fluorescence method for measuring the effective concentration of Mg2+ in the presence of an RNA was used to study Mg2+-RNA interactions with both folded and partially unfolded forms of an RNA pseudoknot. These data established the excess number of Mg2+ ions accumulated by the folded or partially unfolded RNAs as a function of bulk Mg2+ concentration, from which free energies of Mg2+-RNA interactions were derived. Complementary thermal melting experiments were also used to obtain RNA-folding free energies. These experimental data were compared with the results of calculations based on the nonlinear Poisson-Boltzmann equation, which describes the interaction of "diffuse" (fully hydrated) Mg2+ ions with the different RNA forms. Good agreement between the calculations and experimental data suggests that essentially all of the Mg2+-induced stabilization of the native pseudoknot structure arises from the stronger interaction of diffuse ions with the folded tertiary structure compared to that with a partially unfolded state. It is unlikely that the stability of the RNA depends on dehydrated ions bound to specific sets of RNA ligands in the folded state. The data also suggest that the Mg2+-dependent free energy of folding is sensitive to factors that influence the ensemble of RNA conformations present in the partially unfolded state.

Rational selection of small molecules that increase transcription through the GAA repeats found in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive trinucleotide repeat disease with no effective therapy. Expanded GAA repeats in the first intron of the FRDA gene are thought to form unusual non-B DNA conformations that decrease transcription and subsequently reduce levels of the encoded protein, frataxin. Frataxin plays a crucial role in iron metabolism and detoxification. To discover small molecules that increase transcription through the GAA repeat region in FRDA, we have made stable cell lines containing a portion of expanded intron 1 fused to a GFP reporter. Small molecules identified using the competition dialysis method were found to increase FRDA-intron 1-reporter gene expression. One of these compounds, pentamidine, increases frataxin levels in patient cells. Thus our approach can be used to detect small molecules of potential therapeutic value in FRDA.

Conformational changes involved in initiation of minus-strand synthesis of a virus-associated RNA.

Synthesis of wild-type levels of turnip crinkle virus (TCV)-associated satC complementary strands by purified, recombinant TCV RNA-dependent RNA polymerase (RdRp) in vitro was previously determined to require 3' end pairing to the large symmetrical internal loop of a phylogenetically conserved hairpin (H5) located upstream from the hairpin core promoter. However, wild-type satC transcripts, which fold into a single detectable conformation in vitro as determined by temperature-gradient gel electrophoresis, do not contain either the phylogenetically inferred H5 structure or the 3' end/H5 interaction. This implies that conformational changes are required to produce the phylogenetically inferred H5 structure for its pairing with the 3' end, which takes place subsequent to the initial conformation assumed by the RNA and prior to transcription initiation. The DR region, located 140 nucleotides upstream from the 3' end and previously determined to be important for transcription in vitro and replication in vivo, is proposed to have a role in the conformational switch, since stabilizing the phylogenetically inferred H5 structure decreases the negative effects of a DR mutation in vivo. In addition, high levels of aberrant transcription correlate with a specific conformational change in the Pr while maintaining the same conformation of the 3' terminus. These results suggest that a series of events that promote conformational changes is needed to expose the 3' terminus to the RdRp for accurate synthesis of wild-type levels of complementary strands in vitro.

Magnesium dependence of the amplified conformational switch in the trans-acting hepatitis delta virus ribozyme.

The ability of divalent metal ions to participate in both structure formation and catalytic chemistry of RNA enzymes (ribozymes) has made it difficult to separate their cause and effect in ribozyme function. For example, the recently solved crystal structures of precursor and product forms of the cis-cleaving genomic hepatitis delta virus (HDV) ribozyme show a divalent metal ion bound in the active site that is released upon catalysis due to an RNA conformational change. This conformational switch is associated with a repositioning of the catalytically involved base C75 in the active-site cleft, thus controlling catalysis. These findings confirm previous data from fluorescence resonance energy transfer (FRET) on a trans-acting form of the HDV ribozyme that found a global conformational change to accompany catalysis. Here, we further test the conformational switch model by measuring the Mg(2+) dependence of the global conformational change of the trans-acting HDV ribozyme, using circular dichroism and time-resolved FRET as complementary probes of secondary and tertiary structure formation, respectively. We observe significant differences in both structure and Mg(2+) affinity of the precursor and product forms, in the presence and absence of 300 mM Na(+) background. The precursor shortens while the product extends with increasing Mg(2+) concentration, essentially amplifying the structural differences observed in the crystal structures. In addition, the precursor has an approximately 2-fold and approximately 13-fold lower Mg(2+) affinity than the product in secondary and tertiary structure formation, respectively. We also have compared the C75 wild-type with the catalytically inactive C75U mutant and find significant differences in global structure and Mg(2+) affinity for both their precursor and product forms. Significantly, the Mg(2+) affinity of the C75 wild-type is 1.7-2.1-fold lower than that of the C75U mutant, in accord with the notion that C75 is essential for a catalytic conformational change that leads to a decrease in the local divalent metal ion affinity and release of a catalytic metal. Thus, a consistent picture emerges in which divalent metal ions and RNA functional groups are intimately intertwined in affecting structural dynamics and catalysis in the HDV ribozyme.

Kissing complex-mediated dimerisation of HIV-1 RNA: coupling extended duplex formation to ribozyme cleavage.

Initiation of retroviral genomic RNA dimerisation is mediated by the mutual interaction of the dimerisation initiation site (DIS) stem-loops near to the 5' end of the RNA. This process is thought to involve formation of a transient 'kissing' complex over the self-complementary loop bases, which then refolds into a more stable extended interaction. We have developed a novel experimental system that allows us to clearly detect the extended duplex in vitro. Ribozyme sequences were incorporated into or adjacent to the type 1 human immunodeficiency virus DIS stem, leading to the formation of a functional ribozyme only in the extended duplex conformer. Here we show that extended duplex formation results in ribozyme cleavage, thus demonstrating the double-stranded nature of the extended complex and confirming that refolding occurs via melting of the DIS stems. Loop complementarity is essential for extended duplex formation but no sequence requirements for the loops were observed. Efficiency of extended duplex formation is dependent on the strength of the loop-loop interaction, temperature, the magnesium concentration and is strongly accelerated by the viral nucleocapsid protein NCp7. Our ribozyme-coupled approach should be applicable to the analyses of other refolding processes involving RNA loop-loop interactions.

Targeting DNA bulged microenvironments with synthetic agents: lessons from a natural product.

Bulged regions of nucleic acids are important structural motifs whose function has been linked to a number of key nuclear processes. Additionally, bulged intermediates have been implicated in the etiology of several genetic diseases and as targets for viral regulation. Despite these obvious ramifications, few molecules are capable of selective binding to bulged sequences. Prompted by the remarkable affinity of a natural product metabolite, we have designed and prepared a series of readily accessible synthetic agents with selective bulge binding activity. Furthermore, by screening a library of bulge-containing oligodeoxynucelotides, correlations between structure and affinity of the agents can be drawn. In addition to potential applications in molecular biology, the availability of these spirocyclic agents now opens the door for rational drug design.

Magnesium-DNA interactions and the possible relation of magnesium to carcinogenesis. Irradiation and free radicals.

Magnesium deficiency causes renal complications. The appearance of several diseases is related to its depletion in the human body. In radiotherapy, as well as in chemotherapy, especially in treatment of cancers with cis-platinum, hypomagnesaemia is observed. The site effects of chemotherapy that are due to hypomagnesaemia are decreased using Mg supplements. The role of magnesium in DNA stabilization is concentration dependent. At high concentrations there is an accumulation of Mg binding, which induces conformational changes leading to Z-DNA, while at low concentration there is deficiency and destabilization of DNA. The biological and clinical consequences of abnormal concentrations are DNA cleavage leading to diseases and cancer. Carcinogenesis and cell growth are also magnesium-ion concentration dependent. Several reports point out that the interaction of magnesium in the presence of other metal ions showed that there is synergism with Li and Mn, but there is magnesium antagonism in DNA binding with the essential metal ions in the order: Zn>Mg>Ca. In the case of toxic metals such as Cd, Ga and Ni there is also antagonism for DNA binding. It was found from radiolysis of deaerated aqueous solutions of the nucleoside 5'-guanosine monophosphate (5'-GMP) in the presence as well as in the absence of magnesium ions that, although the addition of hydroxyl radicals (*OH) has been increased by 2-fold, the opening of the imidazole ring of the guanine base was prevented. This effect was due to the binding of Mg2+ ions to N7 site of the molecule by stabilizing the five-member ring imitating cis-platinum. It was also observed using Fourier Transform Infrared spectroscopy, Raman spectroscopy and Fast Atom Bombardment mass spectrometry that *OH radicals subtract H atoms from the C1', C4' and C5' sites of the nucleotide. Irradiation of 5'-GMP in the presence of oxygen (2.5 x 10(-4) M) shows that magnesium is released from the complex. There is spectroscopic evidence that superoxide anions (O2-*) react with magnesium ions leading to magnesium release from the complex. From radiolysis data it was suggested that magnesium ions can act as radiosensitizers in the absence of oxygen, while in the presence of oxygen they act as protectors and stabilizers of DNA.

The structural basis for molecular recognition by the vitamin B 12 RNA aptamer.

Previous solution structures of ligand-binding RNA aptamers have shown that molecular recognition is achieved by the folding of an initially unstructured RNA around its cognate ligand, coupling the processes of RNA folding and binding. The 3 A crystal structure of the cyanocobalamin (vitamin B12) aptamer reported here suggests a different approach to molecular recognition in which elements of RNA secondary structure combine to create a solvent-accessible docking surface for a large, complex ligand. Central to this structure is a locally folding RNA triplex, stabilized by a novel three-stranded zipper. Perpendicular stacking of a duplex on this triplex creates a cleft that functions as the vitamin B12 binding site. Complementary packing of hydrophobic surfaces, direct hydrogen bonding and dipolar interactions between the ligand and the RNA appear to contribute to binding. The nature of the interactions that stabilize complex formation and the possible uncoupling of folding and binding for this RNA suggest a strong mechanistic similarity to typical protein-ligand complexes.