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  • Articles  (18)
  • Phsyical and Biochemical Characterisation of DNA  (11)
  • Ribosomes and Protein Translation  (7)
  • Oxford University Press  (18)
  • Copernicus
  • Institute of Physics
  • 2010-2014  (18)
  • 1950-1954
  • Biology  (18)
  • Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
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  • Articles  (18)
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  • Oxford University Press  (18)
  • Copernicus
  • Institute of Physics
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  • Biology  (18)
  • Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
  • 1
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    Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism (2014)
    Oxford University Press
    Details
    Publication Date: 2014-11-07
    Description: 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.
    Keywords: Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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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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    Publication Date: 2014-11-07
    Description: 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.
    Keywords: Phsyical and Biochemical Characterisation of DNA
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    Topics: Biology
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  • 3
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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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    Publication Date: 2014-11-28
    Description: 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.
    Keywords: Phsyical and Biochemical Characterisation of DNA
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    Topics: Biology
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  • 4
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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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    Publication Date: 2012-07-22
    Description: 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.
    Keywords: Phsyical and Biochemical Characterisation of DNA
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    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 5
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    Reconstitution of translation from Thermus thermophilus reveals a minimal set of components sufficient for protein synthesis at high temperatures and functional conservation of modern and ancient translation components (2012)
    Oxford University Press
    Details
    Publication Date: 2012-09-13
    Description: Thermus thermophilus is a thermophilic model organism distantly related to the mesophilic model organism E. coli . We reconstituted protein translation of Thermus thermophilus in vitro from purified ribosomes, transfer ribonucleic acids (tRNAs) and 33 recombinant proteins. This reconstituted system was fully functional, capable of translating natural messenger RNA (mRNA) into active full-length proteins at temperatures up to 65°C and with yields up to 60 μg/ml. Surprisingly, the synthesis of active proteins also occurred at 37°C, a temperature well below the minimal growth temperature for T. thermophilus . A polyamine was required, with tetraamine being most effective, for translation at both high and low temperatures. Using such a defined in vitro system, we demonstrated a minimal set of components that are sufficient for synthesizing active proteins at high temperatures, the functional compatibility of key translation components between T. thermophilus and E. coli , and the functional conservation of a number of resurrected ancient elongation factors. This work sets the stage for future experiments that apply abundant structural information to biochemical characterization of protein translation and folding in T. thermophilus . Because it contains significantly reduced nucleases and proteases, this reconstituted thermostable cell-free protein synthesis system may enable in vitro engineering of proteins with improved thermostability.
    Keywords: Ribosomes and Protein Translation
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  • 6
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    Real-time assay for testing components of protein synthesis (2012)
    Oxford University Press
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    Publication Date: 2012-06-28
    Description: We present a flexible, real-time-coupled transcription–translation assay that involves the continuous monitoring of fluorescent Emerald GFP formation. Along with numerical simulation of a reaction kinetics model, the assay permits quantitative estimation of the effects on full-length protein synthesis of various additions, subtractions or substitutions to the protein synthesis machinery. Since the assay uses continuous fluorescence monitoring, it is much simpler and more rapid than other assays of protein synthesis and is compatible with high-throughput formats. Straightforward alterations of the assay permit determination of (i) the fraction of ribosomes in a cell-free protein synthesis kit that is active in full-length protein synthesis and (ii) the relative activities in supporting protein synthesis of modified (e.g. mutated, fluorescent-labeled) exogenous components (ribosomes, amino acid-specific tRNAs) that replace the corresponding endogenous components. Ribosomes containing fluorescent-labeled L11 and tRNAs labeled with fluorophores in the D-loop retain substantial activity. In the latter case, the extent of activity loss correlates with a combination of steric bulk and hydrophobicity of the fluorophore.
    Keywords: Ribosomes and Protein Translation
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  • 7
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    Dicodon monitoring of protein synthesis (DiCoMPS) reveals levels of synthesis of a viral protein in single cells (2013)
    Oxford University Press
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    Publication Date: 2013-10-08
    Description: The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Förster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the background transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2-Ibaraki and measured, through DiCoMPS, the synthesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fl-tRNA Ile UAU -generated FRET signals were specifically enhanced in infected cells, increased in the course of infection and were diminished on siRNA-mediated knockdown of NS3. Our results establish an experimental approach for the single-cell measurement of the levels of synthesis of a specific viral protein.
    Keywords: Ribosomes and Protein Translation
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  • 8
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    Purification, characterization and crystallization of the human 80S ribosome (2014)
    Oxford University Press
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    Publication Date: 2014-04-03
    Description: Ribosomes are key macromolecular protein synthesis machineries in the cell. Human ribosomes have so far not been studied to atomic resolution because of their particularly complex structure as compared with other eukaryotic or prokaryotic ribosomes, and they are difficult to prepare to high homogeneity, which is a key requisite for high-resolution structural work. We established a purification protocol for human 80S ribosomes isolated from HeLa cells that allows obtaining large quantities of homogenous samples as characterized by biophysical methods using analytical ultracentrifugation and multiangle laser light scattering. Samples prepared under different conditions were characterized by direct single particle imaging using cryo electron microscopy, which helped optimizing the preparation protocol. From a small data set, a 3D reconstruction at subnanometric resolution was obtained showing all prominent structural features of the human ribosome, and revealing a salt concentration dependence of the presence of the exit site tRNA, which we show is critical for obtaining crystals. With these well-characterized samples first human 80S ribosome crystals were obtained from several crystallization conditions in capillaries and sitting drops, which diffract to 26 Å resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future high-resolution work.
    Keywords: Ribosomes and Protein Translation
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    Topics: Biology
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  • 9
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    In vitro experimental system for analysis of transcription-translation coupling (2012)
    Oxford University Press
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    Publication Date: 2012-03-29
    Description: Transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. During transcription–translation, both machineries mutually affect each others’ functions, which is important for regulation of gene expression. Analysis of interactions between RNA polymerase (RNAP) and the ribosome, however, are limited due to the lack of an in vitro experimental system. Here, we report the development of an in vitro transcription coupled to translation system assembled from purified components. The system allows controlled stepwise transcription and simultaneous stepwise translation of the nascent RNA, and permits investigation of the interactions of RNAP with the ribosome, as well as the effects of translation on transcription and transcription on translation. As an example of usage of this experimental system, we uncover complex effects of transcription–translation coupling on pausing of transcription.
    Keywords: Ribosomes and Protein Translation
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    Topics: Biology
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  • 10
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    Translation inhibitors cause abnormalities in ribosome profiling experiments (2014)
    Oxford University Press
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    Publication Date: 2014-09-27
    Description: Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in widespread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods.
    Keywords: Ribosomes and Protein Translation
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    Topics: Biology
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  • 11
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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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    Publication Date: 2014-10-10
    Description: 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.
    Keywords: Phsyical and Biochemical Characterisation of DNA
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    Topics: Biology
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  • 12
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    Probing the elastic limit of DNA bending (2014)
    Oxford University Press
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    Publication Date: 2014-09-17
    Description: 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.
    Keywords: Phsyical and Biochemical Characterisation of DNA
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    Topics: Biology
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  • 13
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    A capture approach for supercoiled plasmid DNA using a triplex-forming oligonucleotide (2013)
    Oxford University Press
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    Publication Date: 2013-05-29
    Description: 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.
    Keywords: Phsyical and Biochemical Characterisation of DNA
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  • 14
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    Sequence-specific electron injection into DNA from an intermolecular electron donor (2013)
    Oxford University Press
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    Publication Date: 2013-04-23
    Description: 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.
    Keywords: Phsyical and Biochemical Characterisation of DNA
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  • 15
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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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    Publication Date: 2013-11-02
    Description: 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.
    Keywords: Phsyical and Biochemical Characterisation of DNA
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  • 16
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    Amplified stretch of bottlebrush-coated DNA in nanofluidic channels (2013)
    Oxford University Press
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    Publication Date: 2013-11-02
    Description: 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.
    Keywords: Phsyical and Biochemical Characterisation of DNA
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  • 17
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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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    Publication Date: 2012-11-25
    Description: 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.
    Keywords: Phsyical and Biochemical Characterisation of DNA
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  • 18
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    Evaluation of TRAP-sequencing technology with a versatile conditional mouse model (2014)
    Oxford University Press
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    Publication Date: 2014-01-28
    Description: Gene expression profiling of various cell lineages has provided invaluable insights into the molecular mechanisms regulating cellular development and differentiation. However, in vivo molecular profiling of rare and interspersed cell populations, such as endothelial cells, has remained challenging. We have generated a versatile floxed translating ribosome affinity purification (TRAP) mouse model, mCherryTRAP, for Cre-dependent translational profiling of distinct cell lineages from intact tissues. To identify cell type–specific transcripts using TRAP, the data have to be filtered to remove both background transcripts not expressed in the profiled cell type and transcripts expressed in all cell populations of the tissue/organ. Filtering has previously been achieved using transcribed RNA from the tissue/organ. Using the mCherryTRAP model, we demonstrate extensive differential expression of RNAs between the translatome and transcriptome of embryonic brains and kidneys. We evaluate the implications of these data for TRAP studies of abundant and rare cell populations. Finally, we demonstrate the applicability of the technology to study organ-specific endothelial cell differentiation.
    Keywords: Ribosomes and Protein Translation
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