Single-stranded Polynucleotides Research Articles

A simple model for exploring conformation of highly-charged electrosprayed single-stranded oligonucleotides

The study of the maximum charge state versus the molecular weight of single-stranded polynucleotides analyzed by electrospray reveals that single strands adopt a compact conformation in positive ion mode, whereas linear structures are predominant in negative ion mode.

A proposition for single molecule DNA sequencing through a nanopore entropic trap

We propose that a DNA entropic trap constructed from a single resizable nanopore could be tuned in real time to capture a single-stranded polynucleotide. With a nanopore of sufficient discriminatory capability, the trapped polynucleotide will be immobilised within the corrugational trough of an inter-nucleotide separation and the prolonged dwell time will enable scrutiny of the nucleotide composition by extended measurement of the resistive pulse signal. The nanopore will be tuned to facilitate single nucleotide translocation, allowing for the processive examination of consecutive nucleotides.

Hyaluronan Production in Synoviocytes as a Consequence of Viral Infections: HAS1 ACTIVATION BY EPSTEIN-BARR VIRUS AND SYNTHETIC DOUBLE- AND SINGLE-STRANDED VIRAL RNA ANALOGS

One of the hallmarks of arthritis is swollen joints containing unusually high quantities of hyaluronan. Intact hyaluronan molecules facilitate cell migration by acting as ligands for CD44. Hyaluronan degradation products, readily formed at sites of inflammation, also fuel inflammatory processes. Irrespective of whether viruses could be a cause of rheumatoid arthritis, there is clear evidence that links viral infections to this debilitating disease. For this study, live Epstein-Barr virus and a number of double- and single-stranded synthetic viral analogs were tested for their effectiveness as activators of hyaluronan (HA) synthesis. As shown herein, Epstein-Barr virus-treated fibroblast-like synoviocytes significantly increase HA production and release. Real time reverse transcription-PCR data show that HAS1 mRNA levels are significantly elevated in virus-treated cells, whereas mRNA levels for the genes HAS2 and HAS3 remain unchanged. As to the mechanism of virus-induced HAS1 transcription, data are presented that imply that among the double- and single-stranded polynucleotides tested, homopolymeric polycytidylic structures are the most potent inducers of HAS1 transcription and HA release, whereas homopolymeric polyinosinic acid is without effect. Analyses of virus-induced signal cascades, utilizing chemical inhibitors of MAPK and overexpressing mutated IKK and IkappaB, revealed that the MAPK p38 as well as the transcription factor NF-kappaB are essential for virus-induced activation of HAS1. The presented data implicate HAS1 as the culprit in unfettered HA release and point out targets in virus-induced signaling pathways that might allow for specific interventions in cases of unwanted and uncontrolled HA synthesis.

Motor step size and ATP coupling efficiency of the dsDNA translocase EcoR124I

The Type I restriction-modification enzyme EcoR124I is an archetypical helicase-based dsDNA translocase that moves unidirectionally along the 3′–5′ strand of intact duplex DNA. Using a combination of ensemble and single-molecule measurements, we provide estimates of two physicochemical constants that are fundamental to a full description of motor protein activity—the ATP coupling efficiency (the number of ATP consumed per base pair) and the step size (the number of base pairs transported per motor step). Our data indicate that EcoR124I makes small steps along the DNA of 1 bp in length with 1 ATP consumed per step, but with some uncoupling of the ATPase and translocase cycles occurring so that the average number of ATP consumed per base pair slightly exceeds unity. Our observations form a framework for understanding energy coupling in a great many other motors that translocate along dsDNA rather than ssDNA.

Viruses as supramolecular self-assemblies: modelling of capsid formation and genome packaging

Viruses are considered the simplest form of live. A virus is basically composed of genomic material surrounded by a protecting capsid. Today, full molecular details of many viruses are known, and several subclasses can be discerned. In this review, we present recent advances made over the past few years obtained from theoretical considerations and model simulations to improve our understanding on two vital aspects of the physics of viruses: viz. viral capsid self-assembly and viral genome packaging. Many processes, such as the self-assembly pathway, genome packing, and ultimately the infection mechanism, differ between viruses containing double-stranded polynucleotides on one hand and single-stranded polynucleotides on the other hand. We believe that these differences to a large degree originate from the different genome flexibilities.

Ionic Current Blockades from DNA and RNA Molecules in the α-Hemolysin Nanopore

We characterize the substate structure of current blockades produced when single-stranded polynucleotide molecules were electrophoretically driven into the α-hemolysin protein pore. We frequently observe substates where the ionic current is reduced by ∼50%. Most of these substates can be associated with a molecular configuration where a polymer occupies only the vestibule region of the pore, though a few appear related to a polymer occupying only the transmembrane β-barrel region of the pore. The duration of the vestibule configuration depends on polymer composition and on which end of the polymer, 3′ or 5′, subsequently threads into the narrowest constriction and initiates translocation. Below ∼140 mV a polymer is more likely to escape from the vestibule against the applied voltage gradient, while at higher voltages a polymer is more likely to follow the voltage gradient by threading through the narrowest constriction and translocating through the pore. Increasing the applied voltage also increases the duration of the vestibule configuration. A semiquantitative model of these trends suggests that escape has stronger voltage dependence than threading, and that threading is sensitive to polymer orientation while escape is not. These results emphasize the utility of α-hemolysin as a model system to study biologically relevant physical and chemical processes at the single-molecule level.

Toward the mechanism of dynamical couplings and translocation in hepatitis C virus NS3 helicase using elastic network model

Hepatitis C virus NS3 helicase is an enzyme that unwinds double-stranded polynucleotides in an ATP-dependent reaction. It provides a promising target for small molecule therapeutic agents against hepatitis C. Design of such drugs requires a thorough understanding of the dynamical nature of the mechanochemical functioning of the helicase. Despite recent progress, the detailed mechanism of the coupling between ATPase activity and helicase activity remains unclear. Based on an elastic network model (ENM), we apply two computational analysis tools to probe the dynamical mechanism underlying the allosteric coupling between ATP binding and polynucleotide binding in this enzyme. The correlation analysis identifies a network of hot-spot residues that dynamically couple the ATP-binding site and the polynucleotide-binding site. Several of these key residues have been found by mutational experiments as functionally important, while our analysis also reveals previously unexplored hot-spot residues that are potential targets for future mutational studies. The conformational changes between different crystal structures of NS3 helicase are found to be dominated by the lowest frequency mode solved from the ENM. This mode corresponds to a hinge motion of the highly flexible domain 2. This motion simultaneously modulates the opening/closing of the domains 1-2 cleft where ATP binds, and the domains 2-3 cleft where the polynucleotide binds. Additionally, a small twisting motion of domain 1, observed in both mode 1 and the computed ATP binding induced conformational change, fine-tunes the binding affinity of the domains 1-3 interface for the polynucleotide. The combination of these motions facilitates the translocation of a single-stranded polynucleotide in an inchworm-like manner.

Electronic Excited States of Polynucleotides: A Study by Electroabsorption Spectroscopy

Electroabsorption spectra were obtained for single-stranded polynucleotides poly(U), poly(C), poly(A), and poly(G) in glycerol/water glass at low temperature, and the differences in permanent dipole moment (Deltamu) and polarizability (Deltaalpha) were estimated for several spectral ranges covering the lowest energy absorption band around 260 nm. In each spectral range, the electrooptical parameters associated with apparent features in the absorption spectrum exhibit distinct values representing either a dominant single transition or the resultant value for a group of a relatively narrow cluster of overlapping transitions. The estimated spacing in energy between electronic origins of these transitions is larger than the electronic coupling within the Coulombic interaction model which is usually adopted in computational studies. The electroabsorption data allow us to distinguish a weak electronic transition associated with a wing in polynucleotide absorption spectra, at an energy below the electronic origin in absorption spectra of monomeric nucleobases. In poly(C) and poly(G), these low-energy transitions are related to increased values of Deltamu and Deltaalpha, possibly indicating a weak involvement of charge resonance in the respective excited states. A model capable of explaining the origin of low-energy excited states, based on the interaction of pipi* and npi* transitions in neighboring bases, is introduced and briefly discussed on the grounds of point dipole interaction.

Stable minihairpin structures forming at minisatellite DNA isolated from yellow fin sea bream Acanthopagrus latus

The lengths of simple repeat sequences are generally unstable or polymorphic (highly variable with respect to the numbers of tandem repeats). Previously we have isolated a family of minisatellite DNA (GenBank accession AF422186) that appears specifically and abundantly in the genome of yellow fin sea bream Acanthopagrus latus but not in closely-related red sea bream Pagrus major, and found that the numbers of tandem arrays in the homologous loci are polymorphic. This means that the minisatellite sequence has appeared and propagated in A. latus genome after speciation. In order to understand what makes the minisatellite widespread within the A. latus genome and what causes the polymorphic nature of the number of tandem repeats, the structural features of single-stranded polynucleotides were analyzed by electrophoresis, chemical modification, circular dichroism (CD), differential scanning calorimetry (DSC) and electron microscopy. The results suggest that a portion of the repeat unit forms a stable minihairpin structure, and it can cause polymerase pausing within the minisatellite DNA.

Selective Photoelectrochemical Detection of DNA with High-Affinity Metallointercalator and Tin Oxide Nanoparticle Electrode

Selective detection of double-stranded DNA (ds-DNA) in solution was achieved by photoelectrochemistry using a high-affinity DNA intercalator, Ru(bpy)2dppz (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) as the signal indicator and tin oxide nanoparticle as electrode material. When Ru(bpy)2dppz alone was irradiated with 470-nm light, anodic photocurrent was detected on the semiconductor electrode due to electron injection from its excited state into the conduction band of the electrode. The current was sustained in the presence of oxalate in solution, which acted as a sacrificial electron donor to regenerate the ground-state metal complex. After addition of double-stranded calf thymus DNA into the solution, photocurrent dropped substantially. The drop was attributed to the intercalation of Ru(bpy)2dppz into DNA and, consequently, the reduced mass diffusion of the indicator to the electrode, as well as electrostatic repulsion between oxalate anion and negative charges on DNA. The degree of signal reduction was a function of the DNA concentration, thus forming the basis for real-time DNA detection. The signal reduction was selective for ds-DNA, as no such effect was observed for single-stranded polynucleotides such as poly-G, poly-C, poly-A, and poly-U. The detection limit of calf thymus ds-DNA reached 1.8 x 10(-10) M in solution.

Substrate Specificity and Structure-Function Analysis of the 3′-Phosphoesterase Component of the Bacterial NHEJ Protein, DNA Ligase D

DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3' end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3'-PO4 or deoxyribonucleoside 3'-PO4 of a primer-template to a 3'-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5' single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2'-OH of the penultimate ribose, it is indifferent to a 2'-OH versus a2'-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2'-OH is moved by 1 nucleotide in the 5' direction, suggesting that LigD is an exoribonuclease that cleaves the 3'-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, Asp15, Glu21, and Glu82, which are critical for 3'-phosphatase activity but not 3'-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3'-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.

Is Hairpin Formation in Single-Stranded Polynucleotide Diffusion-Controlled?

An intriguing puzzle in biopolymer science is the observation that single-stranded DNA and RNA oligomers form hairpin structures on time scales of tens of microseconds, considerably slower than the estimated time for loop formation for a semiflexible polymer of similar length. To address the origin of the slow kinetics and to determine whether hairpin dynamics are diffusion-controlled, the effect of solvent viscosity (eta) on hairpin kinetics was investigated using laser temperature-jump techniques. The viscosity was varied by addition of glycerol, which significantly destabilizes hairpins. A previous study on the viscosity dependence of hairpin dynamics, in which all the changes in the measured rates were attributed to a change in solvent viscosity, reported an apparent scaling of relaxation times (tau(r)) on eta as tau(r) approximately eta(0.8). In this study, we demonstrate that if the effect of viscosity on the measured rates is not deconvoluted from the inevitable effect of change in stability, then separation of tau(r) into opening (tau(o)) and closing (tau(c)) times yields erroneous behavior, with different values (and opposite signs) of the apparent scaling exponents, tau(o) approximately eta(-0.4) and tau(c) approximately eta(1.5). Under isostability conditions, obtained by varying the temperature to compensate for the destabilizing effect of glycerol, both tau(o) and tau(c) scale as approximately eta(1.1+/-0.1). Thus, hairpin dynamics are strongly coupled to solvent viscosity, indicating that diffusion of the polynucleotide chain through the solvent is involved in the rate-determining step.

Phase equilibrium in poly(rA) · poly(rU) complexes with Cd2+ and Mg2+ ions, studied by ultraviolet, infrared, and vibrational circular dichroism spectroscopy

Ultraviolet (UV) and infrared (IR) absorption and vibrational circular dichroism (VCD) spectroscopy were used to study conformational transitions in the double-stranded poly(rA). poly(rU) and its components-single-stranded poly(rA) and poly(rU) in buffer solution (pH 6.5) with 0.1M Na+ and different Mg2+ and Cd2+ (10(-6) to 10(-2) M) concentrations. Transitions were induced by elevated temperature that changed from 10 up to 96 degrees C. IR absorption and VCD spectra in the base-stretching region were obtained for duplex, triplex, and single-stranded forms of poly(rA) . poly(rU) at [Mg2+],[Cd2+]/[P] = 0.3. For single-stranded polynucleotides, the kind of conformational transition (ordering --> disordering --> compaction, aggregation) is conditioned by the dominating type of Me2+-polymer complex that in turn depends on the ion concentration range. The phase diagram obtained for poly(rA) . poly(rU) has a triple point ([Cd2+] approximately 10(-4)M) at which the helix-coil (2 --> 1) transition is replaced with a disproportion transition 2AU --> A2U + poly(rA) (2 --> 3) and the subsequent destruction of the triple helix (3 --> 1). The 2 --> 1 transitions occur in the narrow temperature interval of 2 degrees -5 degrees . Unlike 2 --> 1 and 3 --> 1 melting, the disproportion 2 --> 3 transition is a slightly cooperative one and observed over a wide temperature range. At [Me2+] approximately 10(-3) M, the temperature interval of A2U stability is not less than 20 degrees C. In the case of Cd2+, it increases with the rise of ion concentration due to the decrease of T(m) (2-->3). The T(m) (3-->1) value is practically unchanged up to [Cd2+] approximately 10(-3)M. Differences between diagrams for Mg(2+) and Cd2+ result from the various kinds of ion binding to poly(rA).poly-(rU) and poly(rA).

Resonance Raman and absorption characterization of cationic Co(II)-porphyrin in its complexes with nucleic acids: binding modes, nucleic base specificity and role of water in Co(II) oxidation processes

A comparative study of Co(II) and Co(III) complexes of the water-soluble cationic tetrakis(N-methyl-4-pyridiniumyl)porphyrin [CoII- and CoIIITMpyP(4)] with calf thymus DNA (CT DNA) and synthetic double-stranded (ds) and single-stranded (ss) polynucleotides, namely, poly(dA-dT)2, poly(dG-dC)2, [poly(dA)·poly(dT)], poly(A), poly(dT), poly(G) and poly(C) was carried out by resonance Raman (RR) as well as stationary absorption spectroscopy. It is shown that free CoIITMpyP(4) in aqueous solution is easily oxidized to CoIIITMpyP(4) by dissolved molecular oxygen, but Co(II) to Co(III) oxidation ability fundamentally changes when CoIITMpyP(4) is bound to nucleic acids (NA). In double helical DNA and AT-containing polynucleotides, where the porphyrin is assumed to be outside bound mainly in the minor groove near AT sites, Co(II) is stable for weeks and even months. It is suggested that Co(II) axial ligation by immobilized water (i.e. water molecule that is hydrogen bound to O or N atoms from DNA bases) results in such CoIITMpyP(4) stabilization. In contrast, CoIITMpyP(4) binding with ds and ss guanine-containing polynucleotides results in instantaneous Co(II) to Co(III) transformation. This can be explained by Co ion ligation by the guanine N7 atoms, which are known to be strong electron-donating ligands. It is assumed that partial intercalation occurs when CoIITMpyP(4) is bound with poly(dG-dC)2, in particular, at low ionic strength values, where cobalt ion is Co(III) in character. Copyright © 2005 John Wiley & Sons, Ltd.

Na+ and Mg2+ ion effect on the stability of a poly I . poly A . poly I triple helix.

Differential UV spectroscopy was used to study the temperature dependence of the conformational equilibrium in aqueous poly I . poly A . poly I (A2I) solutions containing Na+ (0.1-2 M) and Mg2+ (10(-5)-0.005 M) ions. Over the whole range of the studied Na+ and Mg2+ concentrations, the heating-induced destruction of the triple A2I helix is actually the A2I --> A + I + I (3 --> 1) transition. The rise of the transition temperature with increasing Na+ and Mg2+ contents is well described by Manning's and the "ligand" theories, which makes it possible to estimate the linear charge density on the single-stranded poly I (xi = 1.9 +/- 0.1) and the Mg2+-A2I binding constant (K = 1,250 M(-1) for the zero degree of binding). An analytical expression has been obtained, which correlates the constants of Mg2+ binding to three- and single-stranded polynucleotides (K3 and K1, respectively) and the linear charge density on them. There are only minor distinctions between the K3 values for A2U and A2I because these polynucleotides have similar structures. The difference in the K1 values is also slight as single-stranded poly U, poly I, and poly A have similar conformations. Dependence of the conformational transition temperatures of two triple helices with changing Mg2+ concentration.

Molecular Weight Control of Curdlan (β-1,3-Glucan Polysaccharide) Provides Unique Polynucleotide Binding Properties

Recently, we found that schizophyllan belonging to a β-1,3-glucan polysaccharide family forms macromolecular complexes with certain single-stranded polynucleotides. The specific complex formation is originated from the β-1,3-glucan skeleton in the main chain. In this paper, we have reported that curdlan, which is a β-1,3-glucan polysaccharide without a side chain glucose unit, shows unique poly(C) binding properties different from those of schizophyllan; in the experiments using curdlan samples with the controlled molecular weight, the complexation ability appeared only in a limited “Mw range window”. Stoichiometric analysis revealed that the “Mw range window” appears as a result of the competition between curdlan–curdlan self-association and curdlan–polynucleotide complexation. Furthermore, when the experiments were extended to other polynucleotides such as poly(A), poly(dA) and poly(dT), the “Mw range window” also appeared at their characteristic molecular weights. We believe, therefore, that curdlan can be applicable, in spite of its cheapness, as a precious functional material to gene technology.

An ecdysone-inducible putative “DEAD box” RNA helicase in the spruce budworm ( Choristoneura fumiferana)

RNA helicases are a family of enzymes that unwind nucleic acid duplexes, such as RNA/RNA and RNA/DNA, in a 3′ to 5′ direction into single-stranded polynucleotides. A putative RNA helicase cDNA ( CfrHlc64) was isolated from the spruce budworm, Choristoneura fumiferana. CfrHlc64 was 1998 nucleotides in length, and the deduced protein had 565 amino acids with a predicted molecular mass of 64 kDa. It contained eight functional motifs conserved in the “DEAD box” family of RNA helicases. The deduced amino acid sequence showed 10–50% identities to homologues of other species from bacteria to human. In vitro expression of the cDNA resulted in recombinant proteins of 64 kDa as expected from the deduced amino acid sequence. Northern blotting and RT-PCR analyses revealed the presence of CfrHlc64 mRNA in all developmental stages from embryo to adult. Higher levels of CfrHlc64 mRNA were detected in the fat body and midgut than in the epidermis of sixth instar larvae. The CfrHlc64 protein was distributed mainly in the fat body. Female adults expressed CfrHlc64 mRNA at higher levels than male adults. The nonsteroidal ecdysone agonist, tebufenozide, enhanced the expression of CfrHlc64 in a dose-dependent manner.

New permanently charged phenanthridinium–nucleobase conjugates. Interactions with nucleotides and polynucleotides and recognition of ds‐polyAH+

AbstractN‐Methyl‐8‐aminophenathridinium–uracil and –adenine conjugates possessing a nucleobase attached at the phenanthridinium 8‐amino group by a trimethylene spacer were prepared in the form of water‐soluble hydrogensulfate salts. Spectroscopic characteristics of the conjugates reveal the formation of folded conformations in water characterized by intramolecular aromatic stacking between the phenanthridinium unit and the tethered nucleobase. The conjugates form 1:1 complexes in water with either complementary or non‐complementary nucleotides, giving log Ks values between 1 and 2 and showing a lack of any base recognition. Also, the binding studies with single‐stranded polynucleotides showed no preference of conjugates to polynucleotides containing complementary nucleobases. At pH 5, the N‐methylphenanthridinium–adenine conjugate exhibited preferred binding to double‐stranded (ds‐) polyAH+, whereas its protonated analogue bound preferably to polyU. The results reveal that the presence of protonated or permanently charged intercalator units in the conjugates dramatically changes their binding preferences for polynucleotides. Copyright © 2003 John Wiley & Sons, Ltd.

Binding of Mg 2+ to single-stranded polynucleotides: hydration and optical studies

The binding of Mg 2+ to single-stranded ribo- and deoxy-polynucleotides, poly(rA), poly(rU), poly(dA) and poly(dT), has been investigated in dilute aqueous solutions at pH 7.5 and 20 °C. A combination of ultrasound velocimetry, density, UV and CD spectroscopy have been employed to study hydration and spectral effects of Mg 2+ binding to the polynucleotides. Volume and compressibility effects of Mg 2+ binding to random-coiled poly(rU) and poly(dT) correspond to two coordination bonds probably between the adjacent phosphate groups. The same parameters for poly(rA)+Mg 2+ correspond to an inner-sphere complex with three–four direct contacts. However, almost no hydration effects are arising in binding to its deoxy analog, poly(dA), indicating mostly a delocalized binding mode. In agreement with hydration studies, optical investigations revealed almost no influence of Mg 2+ on poly(dA) properties, while it stabilizes and aggregates poly(rA) single-helix. The evidence presented here indicates that Mg 2+ are able to bind specifically to single-stranded polynucleotides, and recognize their composition and backbone conformation.

Affinity chromatography of DNA on nonporous copolymerized particles of styrene and glycidyl methacrylate with immobilized polynucleotide

Nonporous particles of microsize were prepared by the dispersion polymerization of styrene and glycidyl methacrylate and chemically modified to introduce amino groups on the surface by grafting with either hexamethylenediamine or N-methyl-1,3-propanediamine. Aminated particles were then coupled with phosphorylated single-stranded polynucleotides at the 5 ′-end through covalent linkages. The affinity columns packed with these prepared polynucleotide-immobilized particles effectively retained single-stranded DNA, which could base-pair with the immobilized sequence. Bound DNAs could be eluted to yield a sharp peak by using an aqueous solution of 0.4 M NaOH. The nonspecific adsorption due to the electrostatic interaction between the polynucleotide and the residual amino groups on the particle surface via the amination with hexamethylenediamine was significant and could only be reduced by using a high salt (NaCl) concentration. A higher salt concentration in the elution solution could result in a portion of complementary polynucleotide eluted in the nonretained fraction. However, the nonspecific adsorption of polynucleotides was insignificant in the column packed with DNA-immobilized particles prepared via amination using N-methyl-1,3-propanediamine. The column was effective for microanalysis of sequence-specific DNA.