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  • Books
  • Articles  (15)
  • Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA  (15)
  • Oxford University Press  (15)
  • MDPI Publishing
  • Nucleic Acids Research  (15)
  • 119207
  • 60967
  • 88336
  • 1
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    Folding complex DNA nanostructures from limited sets of reusable sequences (2016)
    Oxford University Press
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    Publication Date: 2016-06-21
    Description: Scalable production of DNA nanostructures remains a substantial obstacle to realizing new applications of DNA nanotechnology. Typical DNA nanostructures comprise hundreds of DNA oligonucleotide strands, where each unique strand requires a separate synthesis step. New design methods that reduce the strand count for a given shape while maintaining overall size and complexity would be highly beneficial for efficiently producing DNA nanostructures. Here, we report a method for folding a custom template strand by binding individual staple sequences to multiple locations on the template. We built several nanostructures for well-controlled testing of various design rules, and demonstrate folding of a 6-kb template by as few as 10 unique strand sequences binding to 10 ± 2 locations on the template strand.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    Exploded view of higher order G-quadruplex structures through click-chemistry assisted single-molecule mechanical unfolding (2016)
    Oxford University Press
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    Publication Date: 2016-01-09
    Description: Due to the long-range nature of high-order interactions between distal components in a biomolecule, transition dynamics of tertiary structures is often too complex to profile using conventional methods. Inspired by the exploded view in mechanical drawing, here, we used laser tweezers to mechanically dissect high-order DNA structures into two constituting G-quadruplexes in the promoter of the human telomerase reverse transcriptase (hTERT) gene. Assisted with click-chemistry coupling, we sandwiched one G-quadruplex with two dsDNA handles while leaving the other unit free. Mechanical unfolding through these handles revealed transition dynamics of the targeted quadruplex in a native environment, which is named as native mechanical segmentation (NMS). Comparison between unfolding of an NMS construct and that of truncated G-quadruplex constructs revealed a quadruplex–quadruplex interaction with 2 kcal/mol stabilization energy. After mechanically targeting the two G-quadruplexes together, the same interaction was observed during the first unfolding step. The unfolding then proceeded through disrupting the weaker G-quadruplex at the 5'-end, followed by the stronger G-quadruplex at the 3'-end via various intermediates. Such a pecking order in unfolding well reflects the hierarchical nature of nucleic acid structures. With surgery-like precisions, we anticipate this NMS approach offers unprecedented perspective to decipher dynamic transitions in complex biomacromolecules.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 3
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    Conformation-sensitive nucleoside analogues as topology-specific fluorescence turn-on probes for DNA and RNA G-quadruplexes (2015)
    Oxford University Press
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    Publication Date: 2015-12-16
    Description: Development of probes that can discriminate G-quadruplex (GQ) structures and indentify efficient GQ binders on the basis of topology and nucleic acid type is highly desired to advance GQ-directed therapeutic strategies. In this context, we describe the development of minimally perturbing and environment-sensitive pyrimidine nucleoside analogues, based on a 5-(benzofuran-2-yl)uracil core, as topology-specific fluorescence turn-on probes for human telomeric DNA and RNA GQs. The pyrimidine residues of one of the loop regions (TTA) of telomeric DNA and RNA GQ oligonucleotide (ON) sequences were replaced with 5-benzofuran-modified 2'-deoxyuridine and uridine analogues. Depending on the position of modification the fluorescent nucleoside analogues distinguish antiparallel, mixed parallel-antiparallel and parallel stranded DNA and RNA GQ topologies from corresponding duplexes with significant enhancement in fluorescence intensity and quantum yield. Further, these GQ sensors enabled the development of a simple fluorescence binding assay to quantify topology- and nucleic acid-specific binding of small molecule ligands to GQ structures. Together, our results demonstrate that these nucleoside analogues are useful GQ probes, which are anticipated to provide new opportunities to study and discover efficient G-quadruplex binders of therapeutic potential.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
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    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 4
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    Probing a label-free local bend in DNA by single molecule tethered particle motion (2015)
    Oxford University Press
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    Publication Date: 2015-06-24
    Description: Being capable of characterizing DNA local bending is essential to understand thoroughly many biological processes because they involve a local bending of the double helix axis, either intrinsic to the sequence or induced by the binding of proteins. Developing a method to measure DNA bend angles that does not perturb the conformation of the DNA itself or the DNA-protein complex is a challenging task. Here, we propose a joint theory-experiment high-throughput approach to rigorously measure such bend angles using the Tethered Particle Motion (TPM) technique. By carefully modeling the TPM geometry, we propose a simple formula based on a kinked Worm-Like Chain model to extract the bend angle from TPM measurements. Using constructs made of 575 base-pair DNAs with in-phase assemblies of one to seven 6A-tracts, we find that the sequence CA 6 CGG induces a bend angle of 19° ± 4°. Our method is successfully compared to more theoretically complex or experimentally invasive ones such as cyclization, NMR, FRET or AFM. We further apply our procedure to TPM measurements from the literature and demonstrate that the angles of bends induced by proteins, such as Integration Host Factor (IHF) can be reliably evaluated as well.
    Keywords: Nucleic acid structure, 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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    Cooperativity-based modeling of heterotypic DNA nanostructure assembly (2015)
    Oxford University Press
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    Publication Date: 2015-07-25
    Description: DNA origami is a robust method for the fabrication of nanoscale 2D and 3D objects with complex features and geometries. The process of DNA origami folding has been recently studied, however quantitative understanding of it is still elusive. Here, we describe a systematic quantification of the assembly process of DNA nanostructures, focusing on the heterotypic DNA junction—in which arms are unequal—as their basic building block. Using bulk fluorescence studies we tracked this process and identified multiple levels of cooperativity from the arms in a single junction to neighboring junctions in a large DNA origami object, demonstrating that cooperativity is a central underlying mechanism in the process of DNA nanostructure assembly. We show that the assembly of junctions in which the arms are consecutively ordered is more efficient than junctions with randomly-ordered components, with the latter showing assembly through several alternative trajectories as a potential mechanism explaining the lower efficiency. This highlights consecutiveness as a new design consideration that could be implemented in DNA nanotechnology CAD tools to produce more efficient and high-yield designs. Altogether, our experimental findings allowed us to devise a quantitative, cooperativity-based heuristic model for the assembly of DNA nanostructures, which is highly consistent with experimental observations.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
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    Topics: Biology
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  • 6
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    Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules (2015)
    Oxford University Press
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    Publication Date: 2015-03-01
    Description: We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ~15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 7
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    Specific recognition of guanines in non-duplex regions of nucleic acids with potassium tungstate and hydrogen peroxide (2015)
    Oxford University Press
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    Publication Date: 2015-01-10
    Description: Structural features of nucleic acids have become an integral part of current biomedical research. Highly selective and readily performed methods with little toxicity that target guanosines in non-duplex nucleic acids are needed, which led us to search for an effective agent for guanosine sequencing. Treatment of DNA or RNA with potassium tungstate and hydrogen peroxide produced damaged guanosines in DNA or RNA sequences. The damaged guanosines in non-duplex DNA could be cleaved by hot piperidine. Similarly, damaged guanosines in non-duplex RNA could be cleaved by aniline acetate. We could identify structural features of nucleic acid using this strategy instead of dimethyl sulphate and Ribonuclease T1.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
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    Topics: Biology
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  • 8
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    Torsionally constrained DNA for single-molecule assays: an efficient, ligation-free method (2013)
    Oxford University Press
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    Publication Date: 2013-10-19
    Description: Controlled twisting of individual, double-stranded DNA molecules provides a unique method to investigate the enzymes that alter DNA topology. Such twisting requires a single DNA molecule to be torsionally constrained. This constraint is achieved by anchoring the opposite ends of the DNA to two separate surfaces via multiple bonds. The traditional protocol for making such DNA involves a three-way ligation followed by gel purification, a laborious process that often leads to low yield both in the amount of DNA and the fraction of molecules that is torsionally constrained. We developed a simple ligation-free procedure for making torsionally constrained DNA via polymerase chain reaction (PCR). This PCR protocol used two ‘megaprimers’, 400-base-pair long double-stranded DNA that were labelled with either biotin or digoxigenin. We obtained a relatively high yield of gel-purified DNA (~500 ng/100 µl of PCR reaction). The final construct in this PCR-based method contains only one labelled strand in contrast to the traditional construct in which both strands of the DNA are labelled. Nonetheless, we achieved a high yield (84%) of torsionally constrained DNA when measured using an optical-trap-based DNA-overstretching assay. This protocol significantly simplifies the application and adoption of torsionally constrained assays to a wide range of single-molecule systems.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
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    Topics: Biology
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  • 9
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    Heterogeneous staining: a tool for studies of how fluorescent dyes affect the physical properties of DNA (2013)
    Oxford University Press
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    Publication Date: 2013-10-19
    Description: The commonly used fluorescent dye YOYO-1 (YOYO) has, using bulk techniques, been demonstrated to stain DNA heterogeneously at substoichiometric concentrations. We here, using nanofluidic channels and fluorescence microscopy, investigate the heterogeneous staining on the single DNA molecule level and demonstrate that the dye distribution is continuous. The equilibration of YOYO on DNA is extremely slow but can be accelerated by increasing the ionic strength and/or the temperature. Furthermore, we demonstrate how to use the heterogeneous staining as a tool for detailed and time-efficient studies of how fluorescent dyes affect the physical properties of DNA. We show that the relative increase in extension of DNA with increasing amount of YOYO bound is higher at low ionic strengths and also extrapolate the extension of native DNA. Our study reveals important information on how YOYO affects the physical properties of DNA, but it also has broader applications. First, it reveals how cationic intercalators, such as potential DNA drugs, affect DNA under strong confinement. Second, the strategy of using heterogeneous staining is of general use for single molecule studies of DNA interacting with proteins or ligands.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
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    Topics: Biology
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  • 10
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    Purification of DNA-origami nanostructures by rate-zonal centrifugation (2013)
    Oxford University Press
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    Publication Date: 2013-01-20
    Description: Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1–1 µg purified DNA nanostructures, obtaining 〉100 µg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing rate-zonal centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40–80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1–100 µg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
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    Topics: Biology
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  • 11
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    Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome (2013)
    Oxford University Press
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    Publication Date: 2013-02-20
    Description: The mitochondrial genome exists in numerous structural conformations, complicating the study of mitochondrial DNA (mtDNA) metabolism. Here, we describe the development of 2D intact mtDNA agarose gel electrophoresis (2D-IMAGE) for the separation and detection of approximately two-dozen distinct topoisomers. Although the major topoisomers were well conserved across many cell and tissue types, unique differences in certain cells and tissues were also observed. RNase treatment revealed that partially hybridized RNAs associated primarily with covalently closed circular DNA, consistent with this structure being the template for transcription. Circular structures composed of RNA:DNA hybrids contained only heavy-strand DNA sequences, implicating them as lagging-strand replication intermediates. During recovery from replicative arrest, 2D-IMAGE showed changes in both template selection and replication products. These studies suggest that discrete topoisomers are associated with specific mtDNA-directed processes. Because of the increased resolution, 2D-IMAGE has the potential to identify novel mtDNA intermediates involved in replication or transcription, or pathology including oxidative linearization, deletions or depletion.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
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    Topics: Biology
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  • 12
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    Mechanical unfolding of human telomere G-quadruplex DNA probed by integrated fluorescence and magnetic tweezers spectroscopy (2013)
    Oxford University Press
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    Publication Date: 2013-02-20
    Description: Single-molecule techniques facilitate analysis of mechanical transitions within nucleic acids and proteins. Here, we describe an integrated fluorescence and magnetic tweezers instrument that permits detection of nanometer-scale DNA structural rearrangements together with the application of a wide range of stretching forces to individual DNA molecules. We have analyzed the force-dependent equilibrium and rate constants for telomere DNA G-quadruplex (GQ) folding and unfolding, and have determined the location of the transition state barrier along the well-defined DNA-stretching reaction coordinate. Our results reveal the mechanical unfolding pathway of the telomere DNA GQ is characterized by a short distance (〈1 nm) to the transition state for the unfolding reaction. This mechanical unfolding response reflects a critical contribution of long-range interactions to the global stability of the GQ fold, and suggests that telomere-associated proteins need only disrupt a few base pairs to destabilize GQ structures. Comparison of the GQ unfolded state with a single-stranded polyT DNA revealed the unfolded GQ exhibits a compacted non-native conformation reminiscent of the protein molten globule. We expect the capacity to interrogate macromolecular structural transitions with high spatial resolution under conditions of low forces will have broad application in analyses of nucleic acid and protein folding.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
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    Topics: Biology
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  • 13
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    Computational mapping reveals dramatic effect of Hoogsteen breathing on duplex DNA reactivity with formaldehyde (2012)
    Oxford University Press
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    Publication Date: 2012-09-13
    Description: Formaldehyde has long been recognized as a hazardous environmental agent highly reactive with DNA. Recently, it has been realized that due to the activity of histone demethylation enzymes within the cell nucleus, formaldehyde is produced endogenously, in direct vicinity of genomic DNA. Should it lead to extensive DNA damage? We address this question with the aid of a computational mapping method, analogous to X-ray and nuclear magnetic resonance techniques for observing weakly specific interactions of small organic compounds with a macromolecule in order to establish important functional sites. We concentrate on the leading reaction of formaldehyde with free bases: hydroxymethylation of cytosine amino groups. Our results show that in B-DNA, cytosine amino groups are totally inaccessible for the formaldehyde attack. Then, we explore the effect of recently discovered transient flipping of Watson–Crick (WC) pairs into Hoogsteen (HG) pairs (HG breathing). Our results show that the HG base pair formation dramatically affects the accessibility for formaldehyde of cytosine amino nitrogens within WC base pairs adjacent to HG base pairs. The extensive literature on DNA interaction with formaldehyde is analyzed in light of the new findings. The obtained data emphasize the significance of DNA HG breathing.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
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  • 14
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    Label-free, atomic force microscopy-based mapping of DNA intrinsic curvature for the nanoscale comparative analysis of bent duplexes (2012)
    Oxford University Press
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    Publication Date: 2012-06-06
    Description: We propose a method for the characterization of the local intrinsic curvature of adsorbed DNA molecules. It relies on a novel statistical chain descriptor, namely the ensemble averaged product of curvatures for two nanosized segments, symmetrically placed on the contour of atomic force microscopy imaged chains. We demonstrate by theoretical arguments and experimental investigation of representative samples that the fine mapping of the average product along the molecular backbone generates a characteristic pattern of variation that effectively highlights all pairs of DNA tracts with large intrinsic curvature. The centrosymmetric character of the chain descriptor enables targetting strands with unknown orientation. This overcomes a remarkable limitation of the current experimental strategies that estimate curvature maps solely from the trajectories of end-labeled molecules or palindromes. As a consequence our approach paves the way for a reliable, unbiased, label-free comparative analysis of bent duplexes, aimed to detect local conformational changes of physical or biological relevance in large sample numbers. Notably, such an assay is virtually inaccessible to the automated intrinsic curvature computation algorithms proposed so far. We foresee several challenging applications, including the validation of DNA adsorption and bending models by experiments and the discrimination of specimens for genetic screening purposes.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
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  • 15
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    Winding single-molecule double-stranded DNA on a nanometer-sized reel (2012)
    Oxford University Press
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    Publication Date: 2012-10-24
    Description: A molecular system of a nanometer-sized reel was developed from F 1 –ATPase, a rotary motor protein. By combination with magnetic tweezers and optical tweezers, single-molecule double-stranded DNA (dsDNA) was wound around the molecular reel. The bending stiffness of dsDNA was determined from the winding tension (0.9–6.0 pN) and the diameter of the wound loop (21.4–8.5 nm). Our results were in good agreement with the conventional worm-like chain model and a persistence length of 54 ± 9 nm was estimated. This molecular reel system offers a new platform for single-molecule study of micromechanics of sharply bent DNA molecules and is expected to be applicable to the elucidation of the molecular mechanism of DNA-associating proteins on sharply bent DNA strands.
    Keywords: Nucleic acid structure, Phsyical and Biochemical Characterisation of DNA
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    Electronic ISSN: 1362-4962
    Topics: Biology
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