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  • 1
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    Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism (2014)
    Oxford University Press
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    Publikationsdatum: 2014-11-07
    Beschreibung: In recent years, an increasing number of reports have been focused on the structure and biological role of non-canonical nucleic acid secondary structures. Many of these studies involve the use of oligonucleotides that can often adopt a variety of structures depending on the experimental conditions, and hence change the outcome of an assay. The knowledge of the structure(s) formed by oligonucleotides is thus critical to correctly interpret the results, and gain insight into the biological role of these particular sequences. Herein we demonstrate that size-exclusion HPLC (SE-HPLC) is a simple yet surprisingly powerful tool to quickly and effortlessly assess the secondary structure(s) formed by oligonucleotides. For the first time, an extensive calibration and validation of the use of SE-HPLC to confidently detect the presence of different species displaying various structure and/or molecularity, involving 〉110 oligonucleotides forming a variety of secondary structures (antiparallel, parallel, A-tract bent and mismatched duplexes, triplexes, G-quadruplexes and i-motifs, RNA stem loops), is performed. Moreover, we introduce simple metrics that allow the use of SE-HPLC without the need for a tedious calibration work. We show that the remarkable versatility of the method allows to quickly establish the influence of a number of experimental parameters on nucleic acid structuration and to operate on a wide range of oligonucleotide concentrations. Case studies are provided to clearly illustrate the all-terrain capabilities of SE-HPLC for oligonucleotide secondary structure analysis. Finally, this manuscript features a number of important observations contributing to a better understanding of nucleic acid structural polymorphism.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 2
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    Locked nucleic acid oligomers as handles for single molecule manipulation (2014)
    Oxford University Press
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    Publikationsdatum: 2014-11-07
    Beschreibung: Single-molecule manipulation (SMM) techniques use applied force, and measured elastic response, to reveal microscopic physical parameters of individual biomolecules and details of biomolecular interactions. A major hurdle in the application of these techniques is the labeling method needed to immobilize biomolecules on solid supports. A simple, minimally-perturbative labeling strategy would significantly broaden the possible applications of SMM experiments, perhaps even allowing the study of native biomolecular structures. To accomplish this, we investigate the use of functionalized locked nucleic acid (LNA) oligomers as biomolecular handles that permit sequence-specific binding and immobilization of DNA. We find these probes form bonds with DNA with high specificity but with varied stability in response to the direction of applied mechanical force: when loaded in a shear orientation, the bound LNA oligomers were measured to be two orders of magnitude more stable than when loaded in a peeling, or unzipping, orientation. Our results show that LNA provides a simple, stable means to functionalize dsDNA for manipulation. We provide design rules that will facilitate their use in future experiments.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 3
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    Bacteriophage strain typing by rapid single molecule analysis (2015)
    Oxford University Press
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    Publikationsdatum: 2015-10-15
    Beschreibung: Rapid characterization of unknown biological samples is under the focus of many current studies. Here we report a method for screening of biological samples by optical mapping of their DNA. We use a novel, one-step chemo-enzymatic reaction to covalently bind fluorophores to DNA at the four-base recognition sites of a DNA methyltransferase. Due to the diffraction limit of light, the dense distribution of labels results in a continuous fluorescent signal along the DNA. The amplitude modulations (AM) of the fluorescence intensity along the stretched DNA molecules exhibit a unique molecular fingerprint that can be used for identification. We show that this labelling scheme is highly informative, allowing accurate genotyping. We demonstrate the method by labelling the genomes of and T7 bacteriophages, resulting in a consistent, unique AM profile for each genome. These profiles are also successfully used for identification of the phages from a background phage library. Our method may provide a facile route for screening and typing of various organisms and has potential applications in metagenomics studies of various ecosystems.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 4
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    Sequence-specific recognition of DNA minor groove by an NIR-fluorescence switch-on probe and its potential applications (2015)
    Oxford University Press
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    Publikationsdatum: 2015-10-15
    Beschreibung: In molecular biology, understanding the functional and structural aspects of DNA requires sequence-specific DNA binding probes. Especially, sequence-specific fluorescence probes offer the advantage of real-time monitoring of the conformational and structural reorganization of DNA in living cells. Herein, we designed a new class of D2A (one-donor-two-acceptor) near-infrared (NIR) fluorescence switch-on probe named quinone cyanine–dithiazole ( QCy–DT ) based on the distinctive internal charge transfer (ICT) process for minor groove recognition of AT-rich DNA. Interestingly, QCy–DT exhibited strong NIR-fluorescence enhancement in the presence of AT-rich DNA compared to GC-rich and single-stranded DNAs. We show sequence-specific minor groove recognition of QCy–DT for DNA containing 5'-AATT-3' sequence over other variable (A/T)4 sequences and local nucleobase variation study around the 5'- X ( AATT ) Y -3' recognition sequence revealed that X = A and Y = T are the most preferable nucleobases. The live cell imaging studies confirmed mammalian cell permeability, low-toxicity and selective staining capacity of nuclear DNA without requiring RNase treatment. Further, Plasmodium falciparum with an AT-rich genome showed specific uptake with a reasonably low IC 50 value (〈4 µM). The ease of synthesis, large Stokes shift, sequence-specific DNA minor groove recognition with switch-on NIR-fluorescence, photostability and parasite staining with low IC 50 make QCy–DT a potential and commercially viable DNA probe.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 5
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    Target-responsive DNA-capped nanocontainer used for fabricating universal detector and performing logic operations (2014)
    Oxford University Press
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    Publikationsdatum: 2014-11-28
    Beschreibung: Nucleic acids have become a powerful tool in nanotechnology because of their controllable diverse conformational transitions and adaptable higher-order nanostructure. Using single-stranded DNA probes as the pore-caps for various target recognition, here we present an ultrasensitive universal electrochemical detection system based on graphene and mesoporous silica, and achieve sensitivity with all of the major classes of analytes and simultaneously realize DNA logic gate operations. The concept is based on the locking of the pores and preventing the signal-reporter molecules from escape by target-induced the conformational change of the tailored DNA caps. The coupling of ‘waking up’ gatekeeper with highly specific biochemical recognition is an innovative strategy for the detection of various targets, able to compete with classical methods which need expensive instrumentation and sophisticated experimental operations. The present study has introduced a new electrochemical signal amplification concept and also adds a new dimension to the function of graphene-mesoporous materials hybrids as multifunctional nanoscale logic devices. More importantly, the development of this approach would spur further advances in important areas, such as point-of-care diagnostics or detection of specific biological contaminations, and hold promise for use in field analysis.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 6
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    Sizing femtogram amounts of dsDNA by single-molecule counting (2016)
    Oxford University Press
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    Publikationsdatum: 2016-01-30
    Beschreibung: Modern molecular-biology applications raise renewed interest in sizing minute-amounts of DNA. In this work we utilize single-molecule imaging with in situ size calibration to accurately analyze the size and mass distribution of DNA samples. We exploit the correlation between DNA length and its fluorescence intensity after staining in order to assess the length of individual DNA fragments by fluorescence microscopy. Synthetic reference DNA standards are added to the investigated sample before staining and serve as internal size calibrators, supporting a robust assay for accurate DNA sizing. Our results demonstrate the ability to reconstruct the exact length distribution in a complex DNA sample by sizing a subset containing only femtogram amounts of DNA, thus, outperforming microfluidic gel electrophoresis which is the currently accepted gold standard. This assay may find useful applications for genetic analysis where the exact size distribution of DNA molecules is critical and the availability of genetic material is limited.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 7
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    Single-stranded loops as end-label polarity markers for double-stranded linear DNA templates in atomic force microscopy (2012)
    Oxford University Press
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    Publikationsdatum: 2012-07-22
    Beschreibung: Visualization of DNA–protein interactions by atomic force microscopy (AFM) has deepened our understanding of molecular processes such as DNA transcription. Interpretation of systems where more than one protein acts on a single template, however, is complicated by protein molecules migrating along the DNA. Single-molecule AFM imaging experiments can reveal more information if the polarity of the template can be determined. A nucleic acid-based approach to end-labelling is desirable because it does not compromise the sample preparation procedures for biomolecular AFM. Here, we report a method involving oligonucleotide loop-primed synthesis for the end labelling of double-stranded DNA to discriminate the polarity of linear templates at the single-molecule level. Single-stranded oligonucleotide primers were designed to allow loop formation while retaining 3'-single-strand extensions to facilitate primer annealing to the template. Following a DNA polymerase extension, the labelled templates were shown to have the ability to form open promoter complexes on a model nested gene template using two Escherichia coli RNA polymerases in a convergent transcription arrangement. Analysis of the AFM images indicates that the added loops have no effect on the ability of the promoters to recruit RNA polymerase. This labelling strategy is proposed as a generic methodology for end-labelling linear DNA for studying DNA–protein interactions by AFM.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 8
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    Invincible DNA tethers: covalent DNA anchoring for enhanced temporal and force stability in magnetic tweezers experiments (2014)
    Oxford University Press
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    Publikationsdatum: 2014-10-10
    Beschreibung: Magnetic tweezers are a powerful single-molecule technique that allows real-time quantitative investigation of biomolecular processes under applied force. High pulling forces exceeding tens of picoNewtons may be required, e.g. to probe the force range of proteins that actively transcribe or package the genome. Frequently, however, the application of such forces decreases the sample lifetime, hindering data acquisition. To provide experimentally viable sample lifetimes in the face of high pulling forces, we have designed a novel anchoring strategy for DNA in magnetic tweezers. Our approach, which exploits covalent functionalization based on heterobifunctional poly(ethylene glycol) crosslinkers, allows us to strongly tether DNA while simultaneously suppressing undesirable non-specific adhesion. A complete force and lifetime characterization of these covalently anchored DNA-tethers demonstrates that, compared to more commonly employed anchoring strategies, they withstand 3-fold higher pulling forces (up to 150 pN) and exhibit up to 200-fold higher lifetimes (exceeding 24 h at a constant force of 150 pN). This advance makes it possible to apply the full range of biologically relevant force scales to biomolecular processes, and its straightforward implementation should extend its reach to a multitude of applications in the field of single-molecule force spectroscopy.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 9
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    Probing the elastic limit of DNA bending (2014)
    Oxford University Press
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    Publikationsdatum: 2014-09-17
    Beschreibung: Sharp bending of double-stranded DNA (dsDNA) plays an essential role in genome structure and function. However, the elastic limit of dsDNA bending remains controversial. Here, we measured the opening rates of small dsDNA loops with contour lengths ranging between 40 and 200 bp using single-molecule Fluorescence Resonance Energy Transfer. The relationship of loop lifetime to loop size revealed a critical transition in bending stress. Above the critical loop size, the loop lifetime changed with loop size in a manner consistent with elastic bending stress, but below it, became less sensitive to loop size, indicative of softened dsDNA. The critical loop size increased from ~60 bp to ~100 bp with the addition of 5 mM magnesium. We show that our result is in quantitative agreement with the kinkable worm-like chain model, and furthermore, can reproduce previously reported looping probabilities of dsDNA over the range between 50 and 200 bp. Our findings shed new light on the energetics of sharply bent dsDNA.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 10
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    Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy (2016)
    Oxford University Press
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    Publikationsdatum: 2016-04-21
    Beschreibung: Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 11
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    A capture approach for supercoiled plasmid DNA using a triplex-forming oligonucleotide (2013)
    Oxford University Press
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    Publikationsdatum: 2013-05-29
    Beschreibung: Proteins that recognize and bind specific sites in DNA are essential for regulation of numerous biological functions. Such proteins often require a negative supercoiled DNA topology to function correctly. In current research, short linear DNA is often used to study DNA–protein interactions. Although linear DNA can easily be modified, for capture on a surface, its relaxed topology does not accurately resemble the natural situation in which DNA is generally negatively supercoiled. Moreover, specific binding sequences are flanked by large stretches of non-target sequence in vivo . Here, we present a straightforward method for capturing negatively supercoiled plasmid DNA on a streptavidin surface. It relies on the formation of a temporary parallel triplex, using a triple helix forming oligonucleotide containing locked nucleic acid nucleotides. All materials required for this method are commercially available. Lac repressor binding to its operator was used as model system. Although the dissociation constants for both the linear and plasmid-based operator are in the range of 4 nM, the association and dissociation rates of Lac repressor binding to the plasmid-based operator are ~18 times slower than on a linear fragment. This difference underscores the importance of using a physiologically relevant DNA topology for studying DNA–protein interactions.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 12
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    Sequence-specific electron injection into DNA from an intermolecular electron donor (2013)
    Oxford University Press
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    Publikationsdatum: 2013-04-23
    Beschreibung: Electron transfer in DNA has been intensively studied to elucidate its biological roles and for applications in bottom-up DNA nanotechnology. Recently, mechanisms of electron transfer to DNA have been investigated; however, most of the systems designed are intramolecular. Here, we synthesized pyrene-conjugated pyrrole-imidazole polyamides (PPIs) to achieve sequence-specific electron injection into DNA in an intermolecular fashion. Electron injection from PPIs into DNA was detected using 5-bromouracil as an electron acceptor. Twelve different 5-bromouracil-containing oligomers were synthesized to examine the electron-injection ability of PPI. Product analysis demonstrated that the electron transfer from PPIs was localized in a range of 8 bp from the binding site of the PPIs. These results demonstrate that PPIs can be a useful tool for sequence-specific electron injection.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 13
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    Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution (2013)
    Oxford University Press
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    Publikationsdatum: 2013-11-02
    Beschreibung: Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein–DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein–DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 14
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    Amplified stretch of bottlebrush-coated DNA in nanofluidic channels (2013)
    Oxford University Press
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    Publikationsdatum: 2013-11-02
    Beschreibung: The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy. An enhanced stretch along the channel is observed with increased binding of the cationic block of the polypeptide to DNA. A maximum stretch of 85% of the contour length can be achieved inside a channel with a cross-sectional diameter of 200 nm and at a 2-fold excess of polypeptide with respect to DNA charge. With site-specific fluorescence labelling, it is demonstrated that this maximum stretch is sufficient to map large-scale genomic organization. Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA. The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on silica.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 15
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    Fluorescent intercalator displacement replacement (FIDR) assay: determination of relative thermodynamic and kinetic parameters in triplex formation--a case study using triplex-forming LNAs (2012)
    Oxford University Press
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    Publikationsdatum: 2012-11-25
    Beschreibung: Triplex forming oligonucleotides (TFOs) are the most commonly used approach for site-specific targeting of double stranded DNA (dsDNA). Important parameters describing triplex formation include equilibrium binding constants ( K eq ) and association/dissociation rate constants ( k on and k off ). The ‘fluorescent intercalator displacement replacement’ (FIDR) assay is introduced herein as an operationally simple approach toward determination of these parameters for triplexes involving TC-motif TFOs. Briefly described, relative rate constants are determined from fluorescence intensity changes upon: (i) TFO-mediated displacement of pre-intercalated and fluorescent ethidium from dsDNA targets (triplex association) and (ii) Watson–Crick complement-mediated displacement of the TFO and replacement with ethidium (triplex dissociation). The assay is used to characterize triplexes between purine-rich dsDNA targets and TC-motif TFOs modified with six different locked nucleic acid (LNA) monomers, i.e. conventional and C5-alkynyl-functionalized LNA and α-L-LNA pyrimidine monomers. All of the studied monomers increase triplex stability by decreasing the triplex dissociation rate. LNA-modified TFOs form more stable triplexes than α-L-LNA-modified counterparts owing to slower triplex dissociation. Triplexes modified with C5-(3-aminopropyn-1-yl)-LNA-U monomer Z are particularly stable. The study demonstrates that three affinity-enhancing features can be combined into one high-affinity TFO monomer: conformational restriction of the sugar ring, expansion of the pyrimidine -stacking surface and introduction of an exocyclic amine.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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  • 16
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    Directly lighting up RNA G-quadruplexes from test tubes to living human cells (2015)
    Oxford University Press
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    Publikationsdatum: 2015-11-17
    Beschreibung: RNA G-quadruplexes (G4s) are one of the key components of the transcriptome that act as efficient post-transcriptional regulatory elements in living cells. To conduct further studies of the unique biological functions of RNA G4s, techniques need to be developed that can efficiently recognize RNA G4 structures under various conditions, in fixed cells and living cells, as well as in vitro . This paper presents the development of such a method, a new technique using a cyanine dye called CyT, which can detect both canonical and non-canonical RNA G4 structures from test tubes to living human cells. The ability of CyT to distinguish between G4 and nonG4 RNA offers a promising tool for future RNA G4-based biomarker discovery and potential diagnostic applications.
    Schlagwort(e): Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
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