Nucleic acid interaction with VERO cells. A temperature barrier in the interaction pattern.

The interaction of VERO cell monolayers with spin (nitroxide)-(labeled polynucleotides (1(N)n) was examined by electron spin resonance (ESR) spectroscopy at various temperatures. Nitroxide labels covalently linked to (A)n, (dUfl)n, (U)n and (A)n . (U)n were used to monitor the interaction. The VERO cells were grown on small quartz plates with a cell viability of 95% or better and then used directly for the ESR studies. The ESR results indicated that the interaction between VERO cells and spin-labeled nucleic acids is temperature dependent. No temperature dependence was found when VERO cells were in contact with nitroxide radicals which were free in solution or covalently bound to Sepharose 4B. The temperature dependence established with nitroxide-labeled nucleic acids indicates that a temperature barrier must exist between 20 and 26 degrees C for the interaction between nucleic acids and VERO cells; namely, at 26 degrees C or above spin-labeled nucleic acids interact significantly with a VERO cell surface; whereas, at 20 degrees C the ESR signal reports no interaction. It is concluded that a temperature-dependent phase transition of membrane components or cell surface products active at 26 degrees C or above play a key role in the nucleic acid cell surface interaction process.     

Influence of lipid membranes on the conformational transitions of nucleic acids

The conformational transitions of nucleic acids which were enclosed in reverse phase evaporation vesicles (REV) were studied by thermal denaturation with optical recording. Cloned fragments of double-stranded DNA containing 179 base pairs and 187 base pairs, respectively, and polyA.polyU were enclosed in REV with a yield up to every vesicle containing 50 nucleic acid molecules. With the 179 base pairs DNA enclosed in the vesicle from egg lecithin two well resolved helix-coil transitions could be measured; one is very similar in the midpoint-temperature Tm and halfwidth delta T1/2 to the transition of the free nucleic acid, and the other transition occurs stabilized at a 3.5 degrees C higher Tm-value and with a broader delta T1/2, 2.7 degrees C instead of 0.6 degree C. Both transitions are from nucleic acids inside the vesicles. Varying the surface charge of the lipid membrane by adding the negatively charged phosphatidylserine or phosphatidylglycerol, an optimum in the yield of enclosure and a maximum in the increase in Tm (4.5 degrees C) and delta T1/2 (5.5 degrees C instead of 1.0 degrees C) was obtained at 20% phosphatidylserine or phosphatidylglycerol. In vesicles from pure negatively charged lipids no second population of nucleic acids was observed. Qualitatively, similar effects were observed with polyA.polyU. Stabilization and broadening of the second transition is higher for nucleic acids inside vesicles from lipids with unsaturated fatty acids, as dioleoyl-phosphatidylcholine, than with saturated fatty acids, dipalmitoyl-phosphatidylcholine. Stabilization and broadening decrease with increasing ionic strength, whereas the relative contributions of both transitions to the total hypochromicity remain unchanged; the second transition coincides with the first at 90 mM Na+. From the experimental results it was concluded that the interaction of nucleic acids and lipid membranes is mainly of electrostatic nature. The nucleic acids exist inside the vesicles in two populations, one behaving like nucleic acid free in solution and one influenced by the contact with the membrane. All results are in accordance with a model in which the interaction between the nucleic acid and the membrane is in competition with the dipole-dipole interaction inside the membrane surface.     

RIG‐I detects infection with live Listeria by sensing secreted bacterial nucleic acids

Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill‐defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG‐I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG‐I‐dependent IL‐1β‐production and inflammasome activation. The signalling molecule CARD9 contributed to IL‐1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG‐I provides a mechanistic explanation for efficient induction of immunity by live bacteria.     

How do plant virus nucleic acids move through intercellular connections?

In addition to their function in transport of water, ions, small metabolites, and growth factors in normal plant tissue, the plasmodesmata presumably serve as routes for cell-to-cell movement of plant viruses in infected tissue. Virus cell-to-cell spread through plasmodesmata is an active process mediated by specialized virus encoded movement proteins; however, the mechanism by which these proteins operate is not clear. We incorporate recent information on the biochemical properties of plant virus movement proteins and their interaction with plasmodesmata in a model for transport of nucleic acids through plasmodesmatal channels. We propose that only single stranded (ss) nucleic acids can be transported efficiently through plasmodesmata, and that movement proteins function as molecular chaperones for ss nucleic acids to form unfolded movement protein-ss nucleic acid complexes. These complexes are targeted to plasmodesmata. Plasmodesmatal permeability is then increased following interaction with movement protein and the entire movement complex or its nucleic acid component is translocated across the plasmodesmatal channel.     

The ubiquitin-protein ligase activity of Hdm2 is inhibited by nucleic acids

The proto-oncoprotein Hdm2 is a member of the RING finger-type family of ubiquitin-protein ligases E3. The RING finger domain is assumed to mediate the specific interaction of an E3 with its cognate ubiquitin-conjugating enzyme E2, which catalyzes the covalent attachment of ubiquitin to substrate proteins. In addition, the RING finger domain of Hdm2 is involved in Hdm2 homooligomer formation and has the capacity to bind to RNA in a sequence-specific manner. Here we report that interaction with nucleic acids interferes with both Hdm2/Hdm2 complex formation and auto-ubiquitination of Hdm2 in vitro. Furthermore, although binding of Hdm2 to the tumor suppressor p53 is not inhibited by nucleic acids, Hdm2-mediated ubiquitination of p53 is significantly decreased. Taken together, these results provide the first example of an E3 whose activity can be regulated by direct interaction with nucleic acids.     

Nucleic acids binding strategies of small molecules: Lessons from alkaloids

Background

Nucleic acids are now important targets for therapeutic intervention. Alkaloids are an important class of molecules that have myriad therapeutic utility. Isoquinoline and benzophenanthridine alkaloids exhibit multiple pharmacological activities which are often related to their strong nucleic acid binding abilities. Therefore, a review of their interaction aspects with varying nucleic acid structures is essential for rational design and development as therapeutic agents.

锌指结构：最普遍的核酸识别元件

锌指是最大的DNA结合蛋白家庭,是识别DNA最有效、最成功的一种结构元件。其模块性结构特点及与核酸作用的相对简单性,使其成为研究蛋白-核酸相互作用的理想材料,以及人为设计筛选新的核酸结合蛋白的最佳元件。     

Models of interaction between nucleic acids and proteins. Hydrogen bonding of arginine with nucleic acid bases, phosphate groups and carboxylic acids.

Complex formation between the side chain of arginine and nucleic acid bases has been investigated by proton magnetic resonance in dimethylsulfoxide. Simultaneous formation of two hydrogen bonds leads to a selectivity of arginine interaction towards cytosine and guanine. A comparison is made of the interaction of arginine side chain with nucleic acid bases, phosphate and carboxylate anions. It is shown that interaction between carboxylate and arginine is stronger than between phosphate and arginine. These results are discussed with respect to the selective recognition of nucleic acid bases by arginine side chains and by the arginyl-glutamyl ion pair which could form in proteins interacting with nucleic acids.     

Interaction of aminoacridines with nucleic acids. A pulse-radiolysis study.

Pulse radiolysis has been used to study the interaction of aminoacridines with nucleic acids. The data confirm that there are two modes of binding. These are: a weak interaction which has a maximum binding ratio of one site per dye; and a strong binding process effected by both electrostatic and Van der Waals interactions. The limit of this latter, strong binding mode is approximately six sites per dye. The radiation-induced transient absorption spectrum of benzoflavine is characterized by a pronounced bleaching at 440 nm, which is quenched by the addition of nucleic acids. Mechanisms have been proposed for the reactions of both eaq-and .OH with benzoflavine which account both for the observed bleaching of benzoflavine solutions and for the protective effect of nucleic acids. It is proposed that eaq-reacts with benzoflavine to form a stable benzoflavine semiquinone radical and that .OH reacts with subsequent formation of a very stable benzoflavine hydroxycyclohexadienyl radical.     

Interaction of the antineoplastic agent mitoxantrone with double-stranded nucleic acids

The binding of mitoxantrone with double-helical nucleic acids was investigated by the methods of isothermal microcalorimetry, circular dichroism and absorption at the ionic strength mu = 0.11 and 0.011 M NaCl at temperature region of 30 divided by 60 degrees C. The investigation shows, that at mu = 0.11 M NaCl mitoxantrone interacts with double-helical nucleic acids in one way only. For such conditions using spectrophotometric titration data Scatchard plots for the binding of mitoxantrone with double-helical nucleic acids were constructed. The calculations show that the saturation stoichiometry is one mitoxantrone molecule per 2 divided by 3 base pairs DNA and 6 divided by 8 base pairs RNA. The dependence of binding constant from GC-content is observed. It is shown that the binding enthalpy of mitoxantrone with DNA and RNA increases linearly and reaches -(3.0 +/- 0.5) kkal per 1 mol mitoxantrone. It is shown that a binding mitoxantrone with double-helical nucleic acids, besides the intercalation of rings, a determinate contribution in the binding is given also by electrostatic interaction of side chains mitoxantrone with nucleic acids.     

Simulation and modeling of nucleic acid structure, dynamics and interactions

In moving towards the simulation of larger nucleic acid assemblies over longer timescales that include more accurate representations of the environment, we are nearing the end of an era characterized by single nanosecond molecular dynamics simulation of nucleic acids. We are excited by the promise and predictability of the modeling methods, yet remain prudently cautious of sampling and force field limitations. Highlights include the accurate representation of subtle drug-DNA interactions, the detailed study of modified and unusual nucleic acid structures, insight into the influence of dynamics on the structure of DNA, and exploration of the interaction of solvent and ions with nucleic acids.     

Interaction of resveratrol and genistein with nucleic acids

Resveratrol (RES) and genistein (GEN) are the dietary natural products known to possess chemopreventive property and also the ability to repair DNA damage induced by mutagens/carcinogens. It is believed that the therapeutic activity of these compounds could be primarily due to their interaction with nucleic acids but detailed reports are not available. We here explore the interaction of these drugs with nucleic acids considering DNA and RNA as a potential therapeutic target. The interaction of RES and GEN has been analysed in buffered solution with DNA [saline sodium citrate (SSC)] and RNA [tris ethylene diammine tetra acetic acid (TE)] using UV-absorption and Fourier transform infrared (FTIR) spectroscopy. The UV analysis revealed lesser binding affinity with nucleic acids at lower concentration of RES (P/D = 5.00 and 10.00), while at higher drug concentration (P/D = 0.75, 1.00 and 2.50) hyperchromic effect with shift in the lambda(max) is noted for DNA and RNA. A major RES-nucleic acids complexes was observed through base pairs and phosphate backbone groups with K = 35.782 M(-1) and K = 34.25 M(-1) for DNARES and RNA-RES complexes respectively. At various concentrations of GEN (P/D = 0.25, 0.50, 0.75, 1.00 and 2.50) hyperchromicity with shift in the lambda(max) from 260-->263 nm and 260--> 270 nm is observed for DNA-GEN and RNA-GEN complexes respectively. The binding constant (from UV analysis) for GEN-nucleic acids complexes could not be obtained due to GEN absorbance overlap with that of nucleic acids at 260 nm. Nevertheless a detailed analysis with regard to the interaction of these drugs (RES/GEN) with DNA and RNA could feasibly be understood by FTIR.     

Solution spectroscopy of dipicolinic acid interaction with nucleic acids: Role in spore heat resistance

The effect of dipicolinic acid (DPA) or its calcium chelate (CaDPA) on the spectral characteristics of nucleic acids was examined. Dipicolinic acid was found to displace ethidium gromide from DNA; this indicates that it may bind by intercalation. On interaction with DNA, the ultraviolet absorption spectrum revealed downfield shifts and caused progressive diminution in both DNA and dipicolinate chromophores. The strength and type of interaction may be ion-specific but not discriminatory to any type of base pairing. Spectral analysis also indicated that both dipicolinate and calcium dipicolinate bound to different RNA species, although the mechanism of binding was not elucidated. We conclude that the interaction of dipicolinate/ calcium dipicolinate with nucleic acids is a mechanism whereby water can be removed from spore polynucleotides, increasing their stability to denaturation.     

Transport of oligonucleotides across natural and model membranes

Oligo- and polynucleotides can not diffuse through lipid membrane, however they are taken up by eukaryotic cells by endocytosis mediated by the nucleic acid specific receptors. The compounds find some way to escape from endosomes and reach nucleic acids in both cell nucleus and cytoplasm. Oligonucleotides bind to a few cell surface proteins which take part in the virus-cell interaction and in the development of immune response. Interaction of nucleic acids with cell surface proteins may play a role in development of some pathologies. The biological role of this interaction is unclear. Efficient delivery of oligonucleotides into eukaryotic cells can be achieved in some conditions by natural mechanisms and by using artificial carriers-membrane vehicles and cationic polymer micelles.     

Spermine-nucleic acid interactions: a theoretical study

The interaction of spermine with nucleic acids is simulated theoretically using refined semi-empirical energy formulae and an advanced minimization procedure. Various nucleic acids are considered: model homopolymeric DNA's, a dodecamer (CGCGAATTCGCG) of type B-DNA, as well as a transfer RNA, tRNA^Phe. The dominant role of electrostatic potential in determining the preferential binding sites of spermine is demonstrated in each of these cases and the role of counterions, nucleic acid structure, and base-pair sequence is analyzed.     

蛋白质/核酸相互作用研究方法进展

蛋白质和核酸是构成生命体最为重要的两类生物大分子,蛋白质与核酸的相互作用是分子生物学研究的中心问题之一,它是许多生命活动的重要组成部分。研究蛋白质/核酸相互作用近期采用的新技术有:核酸适体技术、生物信息学方法、蛋白质芯片技术以及纳米技术等。本文就这些新的研究方法进行综述。     

Macromolecular mimicry

Some proteins have been shown to mimic the overall shape and structure of nucleic acids. For some of the proteins involved in translating the genetic information into proteins on the ribosome particle, there are indications that such observations of macromolecular mimicry even extend to similarity in interaction with and function on the ribosome. A small number of structural results obtained outside the protein biosynthesis machinery could indicate that the concept of macromolecular mimicry between proteins and nucleic acids is more general. The implications for the function and evolution of protein biosynthesis are discussed.     

Determination of refractive index increment ratios for protein-nucleic acid complexes by surface plasmon resonance

Three nucleic acid-protein complexes of 1:1 stoichiometry were analyzed by surface plasmon resonance on a Biacore biosensor to test whether or not proteins and nucleic acids yielded similar refractive index increments on binding. The expected maximum response in resonance units, (RU(exp))(max), and the observed one, (RU(obs))(max), on saturation of immobilized targets by interacting partners were compared to determine the ratio of (deltan/deltaC)(protein) to (deltan/deltaC)(nucleic acid), where n is the refractive index at the surface and C is the concentration of one partner. Our results suggest that proteins and nucleic acids behave similarly and that the discrepancy between the expected and observed maximum responses for such complexes reflects inaccurate evaluation of the binding responses. Therefore, no correction of the instrument response is required for protein and nucleic acid interaction studies on a Biacore biosensor.     

Interaction of nucleic acids. VI. Interaction of purine with nucleic acids

The interaction of purine with DNA, tRNA, poly A, poly C, and poly A. poly U complex was investigated. In the presence of purine, the nucleic acids in coil form (such as denatured DNA, poly A and poly C in neutral solutions, or tRNA) have lower optical rotations. In addition, hydrodynamic studies indicate that in purine solutions the denatured DNA has a higher viscosity and a decreased sedimentation coefficient. These findings indicate that through interaction with purine, the bases along the poly-nucleotide chain are unstacked and are separated farther from each other, resulting in increased assymmetry (and possibly volume) of the whole polymer. Thus, the de-naturation effect of purine reported previously can be explained by this preferential interaction of purine with the bases of nucleic acids in coil form through a hydrophobic-costacking mechanism. Results from studies on optical rotation and helix-coil transition show that the interaction of purine is greater with poly A than with poly C. The influence of temperature, Mg⁺⁺ concentration, ionic strength, and purine concentration on the effect of purine on nucleic acid conformation has also been investigated. In all these situations the unraveling of nucleic acid conformation occurs at much lower temperatures (20–40°C lower) in the presence of purine (0.2–0.6M).     

A quantitative characterization of interaction between prion protein with nucleic acids

Binding of recombinant prion protein with small highly structured RNAs, prokaryotic and eukaryotic prion protein mRNA pseudoknots, tRNA and polyA has been studied by the change in fluorescence anisotropy of the intrinsic tryptophan groups of the protein. The affinities of these RNAs to the prion protein and the number of sites where the protein binds to the nucleic acids do not vary appreciably although the RNAs have very different compositions and structures. The binding parameters do not depend upon pH of the solution and show a poor co-operativity. The reactants form larger nucleoprotein complexes at pH 5 compared to that at neutral pH. The electrostatic force between the protein and nucleic acids dominates the binding interaction at neutral pH. In contrast, nucleic acid interaction with the incipient nonpolar groups exposed from the structured region of the prion protein dominates the reaction at pH 5. Prion protein of a particular species forms larger complexes with prion protein mRNA pseudoknots of the same species. The structure of the pseudoknots and not their base sequences probably dominates their interaction with prion protein. Possibilities of the conversion of the prion protein to its infectious form in the cytoplasm by nucleic acids have been discussed.