Academic literature on the topic 'Ss(ds-)-DNA'

Can a wide range of complex biochemical behaviour arise from repeated applications of a highly reduced class of interactions? In particular, can the range of DNA manipulations achieved by protein enzymes be simulated via simple DNA hybridization chemistry? In this work, we develop a biochemical system which we call meta-DNA (abbreviated as mDNA), based on strands of DNA as the only component molecules. Various enzymatic manipulations of these mDNA molecules are simulated via toehold-mediated DNA strand displacement reactions. We provide a formal model to describe the required properties and operations of our mDNA, and show that our proposed DNA nanostructures and hybridization reactions provide these properties and functionality. Our meta-nucleotides are designed to form flexible linear assemblies (single-stranded mDNA ( ss mDNA)) analogous to single-stranded DNA. We describe various isothermal hybridization reactions that manipulate our mDNA in powerful ways analogous to DNA–DNA reactions and the action of various enzymes on DNA. These operations on mDNA include (i) hybridization of ss mDNA into a double-stranded mDNA ( ds mDNA) and heat denaturation of a ds mDNA into its component ss mDNA, (ii) strand displacement of one ss mDNA by another, (iii) restriction cuts on the backbones of ss mDNA and ds mDNA, (iv) polymerization reactions that extend ss mDNA on a template to form a complete ds mDNA, (v) synthesis of mDNA sequences via mDNA polymerase chain reaction, (vi) isothermal denaturation of a ds mDNA into its component ss mDNA, and (vii) an isothermal replicator reaction that exponentially amplifies ss mDNA strands and may be modified to allow for mutations.

Transport of maize streak virus (MSV) DNA into the nucleus of host cells is essential for virus replication and the presence of virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculovirus vector system, and also in tobacco protoplasts with a cauliflower mosaic virus (CaMV) 35S promoter-driven transient gene expression vector. Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells. Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein. We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA. To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1-labeled viral ss or ds DNA, was microinjected into maize and tobacco epidermal cells. Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E. coli proteins alone. These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus.

ABSTRACT RAG1 and RAG2 catalyze the first DNA cleavage steps in V(D)J recombination. We demonstrate that the isolated central domain of RAG1 has inherent single-stranded (ss) DNA cleavage activity, which does not require, but is enhanced by, RAG2. The central domain, therefore, contains the active-site residues necessary to perform hydrolysis of the DNA phosphodiester backbone. Furthermore, the catalytic activity of this domain on ss DNA is abolished by addition of the C-terminal domain of RAG1. The inhibitory effects of this latter domain are suppressed on substrates containing double-stranded (ds) DNA. Together, the activities of the reconstituted domains on ss versus mixed ds-ss DNA approximate the activity of intact RAG1 in the presence of RAG2. We propose how the combined actions of the RAG1 domains may function in V(D)J recombination and also in aberrant cleavage reactions that may lead to genomic instability in B and T lymphocytes.

Dipeptide 4 containing two unnatural amino acids, a modified tyrosine and a phenanthridine derivative, was synthesized. Binding of the dipeptide to a series of polynucleotides including ct-DNA, poly A - poly U, poly (dAdT)2, poly dG - poly dC and poly (dGdC)2 was investigated by thermal denaturation experiments, fluorescence spectroscopy and circular dichroism. Thermal denaturation experiments indicated that dipeptide 4 at pH 5.0, when phenanthridine is protonated, stabilizes ds-DNA, whereas it destabilizes ds-RNA. At pH 7.0, when the phenanthridine is not protonated, effects of 4 to the polynucleotide melting temperatures are negligible. At pH 5.0, dipeptide 4 stabilized DNA double helices, and the changes in the CD spectra suggest different modes of binding to ds-DNA, most likely the intercalation to poly dG- poly dC and non-specific binding in grooves of other DNA polynucleotides. At variance to ds-DNA, addition of 4 destabilized ds-RNA against thermal denaturation and CD results suggest that addition of 4 probably induced dissociation of ds-RNA into ss-RNA strands due to preferred binding to ss-RNA. Thus, 4 is among very rare small molecules that stabilize ds-DNA but destabilize ds-RNA. However, fluorescence titrations with all polynucleotides at both pH values gave similar binding affinity (log Ka ≈ 5), indicating nonselective binding. Preliminary photochemical experiments suggest that dipeptide 4 reacts in the photochemical reaction, which affects polynucleotides chirality, presumably via quinone methide intermediates that alkylate DNA.

ABSTRACT Adeno-associated virus (AAV) is a unique gene transfer vector which takes approximately 4 to 6 weeks to reach its expression plateau. The mechanism for this slow-rise expression profile was proposed to be inefficient second-strand DNA synthesis from the input single-stranded (ss) DNA viral genome. In order to clarify the status of ss AAV genomes, we generated AAV vectors labeled with bromodeoxyuridine (BrdU), a nucleotide analog that can be incorporated into the AAV genome and packaged into infectious virions. Since BrdU-DNA can be detected only by an anti-BrdU antibody when DNA is in an ss form, not in a double-stranded (ds) form, ss AAV genomes with BrdU can be readily tracked in situ. Although ss AAV DNA was abundant by Southern blot analysis, free ss AAV genomes were not detectable after AAV transduction by this new detection method. Further Southern blot analysis of viral DNA and virions revealed that ss AAV DNA was protected within virions. Extracted cellular fractions demonstrated that viral particles in host cells remained infectious. In addition, a significant amount of AAV genomes was degraded after AAV transduction. Therefore, we conclude that the amount of free ss DNA is not abundant during AAV transduction. AAV transduction is limited by the steps that affect AAV ss DNA release (i.e., uncoating) before second-strand DNA synthesis can occur. AAV ss DNA released from viral uncoating is either converted into ds DNA efficiently or degraded by cellular DNA repair mechanisms as damaged DNA. This study elucidates a mechanism that can be exploited to develop new strategies to improve AAV vector transduction efficiency.

Lymphocytes from normal, non-smoking human individuals not taking drugs were isolated from the peripheral blood by means of the lymphoprep method. The cells were cultured in RPMI medium with 10% fetal calf serum and stimulated with Phytohemagglutinin. A mutagen such as 3-methylcholanthrene was added for varying periods of time. Then the subspecies of DNA, i.e. double and single stranded DNA (ds-DNA and ss-DNA), were separated by the alkaline elution technique and quantitated by fluorimetric estimation. The mutagen induced a significant rise in the level of ss-DNA, but no changes in ds-DNA could be traced. The time-dependent changes increased for at least four days of exposure, indicating that the repair enzymes were not able to compensate for the DNA damage.

Abstract RecBCD enzyme of Escherichia coli is required for the major pathway of homologous recombination following conjugation. The enzyme has an ATP-dependent DNA unwinding activity, ATP-dependent single-stranded (ss) and double-stranded (ds) DNA exonuclease activities, and an activity that makes a ss DNA endonucleolytic cut near Chi sites. We have isolated and characterized ten mutations that reduced recombination proficiency and inactivated some, but not all, activities of RecBCD enzyme. One class of mutants had weak ds DNA exonuclease activity and lacked Chi-dependent DNA cleavage activity, a second class lacked only Chi-dependent DNA cleavage activity, and a third class retained all activities tested. The properties of these mutants indicate that the DNA unwinding and ss DNA exonuclease activities of the RecBCD enzyme are not sufficient for recombination. Furthermore, they suggest that the Chi-dependent DNA cleavage activity or another, as yet unidentified activity or both are required for recombination. The roles of the RecBCD enzymatic activities in recombination and exclusion of foreign DNA are discussed in light of the properties of these and other recBCD mutations.

We investigated the virucidal effects in solution of a new type of disinfectant, calcium bicarbonate mesoscopic crystals, designated CAC-717, against various types of virus. CAC-717 in solution is alkaline (pH 12.4) and has a self-electromotive force that generates pulsed electrical fields. Upon application to human skin, the pH of the solution becomes 8.4. CAC-717 contains no harmful chemicals and is thus non-irritating and harmless to humans and animals. Its virucidal effects were tested against six types of animal virus: enveloped double-strand (ds)-DNA viruses, non-enveloped ds-DNA viruses, non-enveloped single strand (ss)-DNA viruses, enveloped ss-RNA viruses, non-enveloped ss-RNA viruses, and non-enveloped ds-RNA viruses. The treatment resulted in a reduction in viral titer of at least 3.00 log10 to 6.38 log10. Fetal bovine serum was added as a representative organic substance. When its concentration was ≥20%, the virucidal effect of CAC-717 was reduced. Real-time PCR revealed that CAC-717 did not reduce the quantity of genomic DNA of most of the DNA viruses, but it greatly reduced that of the genomic RNA of most of the RNA viruses. CAC-717 may therefore be a useful biosafe disinfectant for use against a broad range of viruses.

2013/2014
Advanced nanotechnologies allow the manipulation of molecules with nanoscale precision, and can be used for the production of sensitive devices for protein or nucleic acids detection for clinical use. DNA nano-assemblies are an excellent route for ultrasensitive DNA/RNA detection and for DNA-protein conjugated immobilization, for bio interaction studies, through the careful detection of single strand DNA (ssDNA) hybridization with complementary target sequences. For DNA nanoscale devices, the control of DNA surface density and conformation is crucial in order to achieve the highest reproducibility and to optimize the sensitivity. An improved understanding of the chemical and physical properties of the nanoscale DNA assemblies and of the recognition process is necessary for device performance optimization. In this framework, we first focused on the understanding of the mechanisms that optimize and limit hybridization efficiency in variable density DNA monolayers. We performed Atomic Force Microscopy (AFM) assisted-Nanografting and AFM measurements to realize reference patches into a DNA self-assembled monolayer, and to carefully monitoring DNA hybridization. We then performed molecular dynamics (MD) simulations, in collaboration with a theoretical group, to capture the energetic hybridization limit in high dense DNA monolayers. We found that no more than 44% of the substrate ssDNA can be successfully hybridized, limited by molecular and electrostatic crowding effect connected to the highly charged nature of DNA. To further capture the conformational properties of DNA monolayers, and their relation to biorecognition, we characterized the ionic strength effect on ssDNA nano-assembled of different density by careful AFM topography measurements in liquid environment. We confined ssDNA brushes with controlled surface densities within a bio-repellent self-assembled monolayer. We then monitored the topographic brush height variation upon changing salt type (NaCl, KCl, CaCl2 and MgCl2 ) and concentration inside the liquid cell. We showed that the measured height is related to scaling law of salt concentration, in agreement with the theory of polyelectrolyte brush. Using this scaling model to fit our experimental data, we quantified structural parameters such as the average internucleotide distance (d) for ssDNA brushes of different, estimated surface density σ, featuring a strong dependence of d on different salts species. This result is crucial for the structural designing of synthetic nucleic acids and, more generally, nucleic acid-based devices with controlled physical behaviors. In the last part of the work, we apply all knowledge learned on hybridization mechanism to a clinical problem. We studied the hybridization mechanism to distinguish single base mismatch and to detect at high sensitivity, without any labeling and amplification, microRNAs (miRNAs) connected to hearth failure disease. Our results demonstrate that the AFM nanolithography can serve as a sensitive and selective readout system to discriminate single nucleotide polymorphism. Also, our device allows for the detection of more than one sequence of miRNAs on a same assay with target in picomolar (100pM) range concentration.
I recenti sviluppi delle nanotecnologie permettono di manipolare singole molecole con precisione nanometrica, e possono essere utilizzati per la produzione di dispositivi innovativi ad alta sensitivita` per la rivelazione di proteine e acidi nucleici, per usi clinici. Nanostrutture di DNA a singolo filamento rappresentano una eccellente soluzione per la rivelazione ultrasensibile di frammenti di DNA/RNA e per l’immobilizzazione di coniugati DNA-proteina per studi di bioriconoscimento, attaverso lo studio dell’ibridazione del DNA con le sequenze target complementari. Nello sviluppo di dipositivi alle nanoscale basati sul DNA, il controllo di parametri quali la densita` di superficie e la conformazione del DNA, risulta cruciale per raggiungere gli alti livelli di riproducibilita` richiesti e per ottimizzare la sensitivita`. Studiare e capire in dettaglio le proprieta` chimico -fisiche di strutture alle nanoscale di DNA a singolo filamento, e del relativo processo di bioriconoscimento risulta quindi fondamentale per ottimizzare le prestazioni del dispositivo associato. In questo contesto, ci siamo dapprima focalizzati sullo studio dei meccanismi che ottimizzano e limitano l’efficienza di ibridazione in monolayer di DNA. Usando il microscopio a forza atomica (AFM) e una tecnica di nanolitografia basata sull’AFM, il nanografting, abbiamo costruito delle nanostrutture di riferimento in film di DNA autoassemblati, ad alta densita`, ed abbiamo accuratamente monitorato con l’AFM e con simulazioni di dinamica molecolare, il limite di ibridazione in tali film. In collaborazione con un gruppo di fisici teorici, abbiamo trovato un limite di ibridazione pari a circa il 44% delle sequenze probe, collegandolo a effetti di repulsione elettrostatica dovuta all’ alta densita` a di carica nei monolayer di DNA, un polielettrolita altamente carico in soluzione. In un secondo tempo, per cogliere le proprieta` conformazionali dei monolayer di DNA, e la loro relazione con la capacita` di bioriconoscimento, abbiamo creato delle nanostrutture di DNA a singolo filamento, a densit variabile, in un monostrato autoassemblato di molecole bio-repellenti, e caratterizzato l’effetto della forza ionica della soluzione a mezzo di misure topografiche fatte con l’ AFM, in liquido. Da misure di variazione dell’ altezza topografica delle nanostrutture di DNA in funzione dei diversi sali usati in soluzione (NaCl, KCl, CaCl2 and MgCl2 ) e della loro concentrazione, abbiamo dimostrato che, per ogni sale, l’ altezza` legata alla concentrazione da una legge di scala, in accordo con la teoria dei polyelectrolyte brush. Utilizzando questa legge di scala, abbiamo fatto un fit dei dati sperimentali, quantificando un importante parametro strutturale, la distanza media tra nucleotidi nel filamento (d), per nanostrutture di DNA con divesra densita`, anch’essa stimata dal nostro fit. Questo risultato e` fondamentale per il disegno di acidi nucleici sintetici e piu` in generale per la progettazione di dispositivi miniaturizzati per la rivelazione di acidi nucleici. Nella parte finale di questo lavoro di tesi, abbiamo applicato le conoscenze acquisite sui meccanismi di ibridazione del DNA su scale nanometriche, per realizzare dispositivi utili a scopi clinici. Abbiamo studiato il meccanismo di ibridazione per distinguere un mismatch tra due filamenti complementari di DNA relativo a una singola base e alla rivelazione di micro-RNA, biomarcatori rilevanti per monitorare specifiche malattie quali, nel presente caso, malattie cardiovascolari. Abbiamo dimostrato che i nostri nanodispositivi dimostrano un’ottima risoluzione (100 pM o meglio) e che possono essere utilizzati senza bisogno di amplificazione del materiale genetico originale, o di altre modificazioni, in estratti provenienti da plasmi umani. Queste piattaforme possono essere ulteriormente sviluppate per il monitoraggio di polimorfismi di singolo nucleotide, estremamente rilevanti dal punto di vista clinico
XXVII Ciclo
1986