http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_10 Footprinting is a method for determining the sequence selectivity of DNA-binding compounds in which ligands protect DNA from cleavage at their binding sites. Footprinting templates are typically 50-200 base pairs long, and DNase I is the most commonly used nuclease for these experiments. This chapter describes the preparation and labelling of suitable DNA footprinting substrates, the footprinting experiment itself, and the way in which these data can be used to estimate the dissociation constant of the interaction. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_11 Eukaryotic DNA forms a complex with an equal mass of proteins to form chromatin. To fully understand the action of DNA-reactive antitumor antibiotics in the cell, their effect must be studied in a chromatin context. In particular, it is of interest to investigate how the distortion of DNA, in the context of a nucleosome, affects the action of drugs with either monoalkylation or crosslinking activity, and how modified DNA is assembled into chromatin. Here, we present experimental approaches that allow one to compare the effect of such drugs on free DNA and nucleosomes. We find significant differences that likely arise from the different geometry of nucleosomal DNA compared to free DNA and also find that drug-mediated DNA crosslinking affects nucleosome assembly. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_12 Many DNA-binding small molecules, typically those with a molecular mass less than 1,000 g/mol, recognize duplex DNA with some degree of sequence specificity. These include drugs used to treat several human diseases, including viral and bacterial infections, malaria, and cancer. Determining the binding specificity of DNA-binding molecules can be important for their development, especially if they are being designed to target specific DNA sequences. A limited amount of information can be obtained through the study of small molecule binding to defined naturally occurring or synthetic DNA sequences; however, a full picture of a small molecule’s binding specificity can only be obtained through combinatorial means, whereby vast libraries of sequences are screened. Several combinatorial methods have been developed for the study of ligand-DNA interactions, but only one method, Restriction Endonuclease Protection Selection and Amplification (REPSA), is generally applicable to the study of native small molecule-DNA complexes under physiologic conditions. REPSA may be used with both covalent and noncovalent small molecule-DNA complexes and with mixtures of small molecules with relatively unknown identities and properties. Thus, REPSA is a powerful, versatile, general method for the combinatorial determination of small molecule-DNA binding specificity and a functional means for drug discovery and characterization. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_13 A major class of anticancer agents in current clinical use exerts its anticancer effects by binding covalently or non-covalently to DNA. A detailed understanding of the nature of these drug-DNA complexes would be expected to lead to better uses of these drugs, and also assist with the design of improved drug derivatives. Here, we present a transcriptional footprinting assay that can be implemented to define the DNA sequence specificity and kinetics associated with drug-DNA complexes. The basic steps involve the formation of drug-DNA complexes, the formation of synchronised initiated transcripts, and finally transcriptional elongation to reveal drug blockage sites that impede the progression of RNA polymerase. We have used the “in vitro transcription assay” to investigate many covalent drug-DNA interactions; most notably those obtained using anthracycline anticancer agents such as doxorubicin and anthracenedione-based anticancer agents, including mitoxantrone and pixantrone. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_14 Polypurine/polypyrimidine (pPu/pPy) tracts, which exist in the promoter regions of many growth-related genes, have been proposed to be very dynamic in their conformation. In this chapter, we describe a detailed protocol for DNase I and S1 nuclease footprinting experiments with supercoiled plasmid DNA containing the promoter regions to probe whether there are conformational transitions to B-type DNA, melted DNA, and G-quadruplex structures within this tract. This is demonstrated with the proximal promoter region of the human vascular endothelial growth factor (VEGF) gene, which also contains multiple binding sites for Sp1 and Egr-1 transcription factors. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_15 Several biochemical and biophysical methods are available to study the intercalation of a small molecule between two consecutive base pairs of DNA. Among them, the topoisomerase I-mediated DNA relaxation assay has proved highly efficient, relatively easy to handle and very informative to investigate drug binding to DNA. The test relies on the use of a supercoiled plasmid to mimic the topological constraints of genomic DNA. The three main components of the assay - the topoisomerase I enzyme, DNA helix and intercalating small molecules - are presented here in a structural context. The principle of the assay is described in detail, along with a typical experimental protocol. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_16 We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of topoisomerase enzymes that is also capable of monitoring the activity of other enzymes that alter the topology of DNA. The assay utilises intermolecular triplex formation to resolve supercoiled and relaxed forms of DNA, the principle being the greater efficiency of a negatively supercoiled plasmid to form an intermolecular triplex with an immobilised oligonucleotide than the relaxed form. The assay provides a number of advantages over the standard gel-based methods, including greater speed of analysis, reduced sample handling, better quantitation and improved reliability and accuracy of output data. The assay is performed in microtitre plates and can be adapted to high-throughput screening of libraries of potential inhibitors of topoisomerases including bacterial DNA gyrase. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_17 The Single Cell Gel Electrophoresis (Comet) assay, originally developed to allow visualisation of DNA strand break damage in individual cells, has been adapted to measure DNA interstrand cross-links. DNA interstrand cross-links are formed in cells by a number of commonly used cancer chemotherapy agents and are considered to be the critical lesion formed by such agents. This technique allows the analysis of DNA interstrand cross-link formation and repair at a single cell level, requires few cells, allows the determination of heterogeneity of response within a cell population and is sensitive enough to measure DNA interstrand cross-links at pharmacologically relevant doses. The method can be applied to any in vitro or in vivo application where a single cell suspension can be obtained. The method has also become invaluable in studies using human tissue and can be used as a method for pharmacodynamic analysis in early clinical trials. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_18 Bifunctional DNA damaging agents continue to be the mainstay in various chemotherapeutic regimens used in the clinic. DNA interstrand crosslinks are considered to be the critical cytotoxic lesions for the biological activity of such agents. Gel-based electrophoretic assays can efficiently separate denatured single-stranded DNA from double-stranded, covalently-linked DNA resulting from the presence of an interstrand crosslink. The methods described here offer a simple way for the assessment of crosslinking efficiencies of bifunctional agents in both long fragments of DNA (e.g. 1-5 kb) and short oligonucleotide DNA duplexes. As the repair of interstrand crosslinks is a key determinant of cellular and clinical chemosensitivity, these methods can be useful for the characterization and isolation of site-directed adducted substrates for use in subsequent biochemical analysis of cellular recognition and DNA repair processes. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_19 The targeting of telomerase and telomere maintenance in human cancer cells can be achieved by small molecules that induce the 3'single-stranded ends of telomeric DNA to fold up into four-stranded quadruplex structures that inhibit the action of the telomerase enzyme complex. In this chapter, we describe a series of biochemical, biophysical, and cellular assays that are used to evaluate the activity of new compounds, and so assess whether they are suitable for examination in xenograft models of human cancer. These assays evaluate quadruplex stabilisation properties, short- and long-term cell viability, telomerase enzymatic activity, cellular senescence, and telomere length changes. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_1 Surface plasmon resonance (SPR) technology with biosensor surfaces has become a widely-used tool for the study of nucleic acid interactions without any labeling requirements. The method provides simultaneous kinetic and equilibrium characterization of the interactions of biomolecules as well as small molecule-biopolymer binding. SPR monitors molecular interactions in real time and provides significant advantages over optical or calorimetic methods for systems with strong binding coupled to small spectroscopic signals and/or reaction heats. A detailed and practical guide for nucleic acid interaction analysis using SPR-biosensor methods is presented. Details of the SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips, and samples, as well as extensive information on experimental design, quantitative and qualitative data analysis and presentation. A specific example of the interaction of a minor-groove-binding agent with DNA is evaluated by both kinetic and steady-state SPR methods to illustrate the technique. Since the molecules that bind cooperatively to specific DNA sequences are attractive for many applications, a cooperative small molecule-DNA interaction is also presented. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_2 A simple thermal melting experiment may be used to demonstrate the stabilization of a given structure by a ligand (usually a small molecule, sometimes a peptide). Preparation of the sample is straightforward, and the experiment itself requires an inexpensive apparatus. Furthermore, reasonably low amounts of sample are required. A qualitative analysis of the data is simple: An increase in the melting temperature (T m) indicates preferential binding to the folded form as compared to the unfolded form. However, it is perilous to derive an affinity constant from an increase in T m as other factors play a role. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_3 When a drug binds to DNA, its electronic structure is perturbed, and it perturbs the DNA’s electronic structure. The resulting change to the electronic spectroscopy can be used to probe the drug-DNA interaction. This chapter outlines how circular and linear dichroism spectroscopy can be used to provide information about drug-DNA systems. Circular dichroism spectroscopy involves measuring the difference in absorption of left and right circularly polarized light. It is uniquely sensitive to the helicity of the molecules being studied. Linear dichroism, as the name implies, involves measuring the difference in absorption of light linearly polarized parallel and perpendicular to an orientation axis. Linear dichroism provides information about the relative orientations of subunits of an interacting system. The material presented in this chapter is by no means comprehensive; the aim is to enable the user to collect reasonable quality data and to interpret it. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_4 DNA·RNA hybrid duplexes are functionally important structures in gene expression that are underutilized as potential drug targets. Several tools are described here for the discovery and characterization of small molecules capable of the selective recognition of DNA·RNA hybrid structures. Competition dialysis and thermal denaturation of mixtures of polynucleotide structures can be used to identify small molecules that bind selectively to DNA·RNA hybrids. An assay that measures small molecule inhibition of RNase H can be used to measure a functional response to these ligands. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_5 The design of new DNA-targeted molecules, primarily for use in the therapy of diseases such as cancer, relies on the assessment of both affinity for DNA and selectivity of binding to choosen base pair sequences. Capillary electrophoresis, with a polymer added to the running buffer, is very well suited to the separation of oligonucleotides in the range 12-20 base pairs, with the separation based on length rather than base pair sequence. In this way, it is possible to conduct competition experiments using mixtures of up to four oligonucleotides and giving a direct measure of the relative affinity of high-affinity ligands, specifically those binding in the minor groove with slow on-off rates. The relative affinities can be securely quantified, even where the affinities are very high. Working from first principles, it is shown that the measurement of absolute affinities presents various problems, not least that the concentration of DNA and ligand used in the experiment will affect the magnitude of K d, which is not constant. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_6 Electrospray mass spectrometry can be used to detect ligand-DNA noncovalent complexes formed in solution. This chapter describes how to determine equilibrium association constants of the complexes. Particular attention is devoted to describing how to tune an electrospray mass spectrometer using a 12-mer oligodeoxynucleotides duplex in order to perform these experiments. This protocol can then be applied to any nucleic acid structure that can be ionized with electrospray mass spectrometry. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_7 There have been many attempts in the past to determine whether significant levels of Adriamycin-DNA adducts form in cells and contribute to the anticancer activity of this agent. Supraclincal drug levels have been required to study drug-DNA adducts because of the lack of sensitivity associated with many of the techniques employed, including liquid scintillation counting of radiolabeled drug. The use of accelerator mass spectrometry (AMS) has provided the first direct evidence of Adriamycin-DNA adduct formation in cells at clinically relevant Adriamycin concentrations. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin-DNA adduct detection (compared to liquid scintillation counting) and has revealed adduct formation within an hour of drug treatment. The rigorous protocol required for this approach, together with many notes on the precautions and procedures required in order to ensure that absolute levels of Adriamycin-DNA adducts can be determined with good reproducibility, is outlined in this chapter. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_8 We describe a molecular modelling method for calculating the binding affinity of ligands for DNA. Though theoretically applicable to any form of noncovalent interaction, we concentrate on the case of predicting the sequence selectivity of a minor-groove binding ligand. The method is based on performing molecular dynamics (MD) simulations on DNA sequences, with and without the ligand bound, and postprocessing the molecular dynamics trajectory data to obtain approximate free energies of binding. We discuss issues relating to the preparation of the structures for simulation, choices for the molecular dynamics simulation method itself, methods for evaluating the reliability and stability of the simulation data, and finally alternative approaches to postprocessing the data to extract approximate free energies of binding. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-418-0_9 Anomalous scattering is commonly used to solve X-ray structures. As discussed here, anomalous scattering is also useful for characterizing complex systems with mixed and partial occupancies, where true electron density is represented by unresolvable ensemble averages. The solvent environment surrounding nucleic acids is an example of such a system, as are some DNA-ligand systems. The atomic number and wavelength dependencies of anomalous scattering allow one to filter out the electron densities of C, N, and O, and to cleanly visualize the electron densities of heavier atoms. Therefore, anomalous scattering can make beacons of selected atoms. In addition, anomalous scattering provides a model-independent method for determining atomic identities. Here, we describe applications of anomalous scattering to the structure determination of DNA-platinum complexes and in cation associations of free DNA, of DNA-anthracycline complexes, of chemically modified DNA, and of DNA-protein complexes. The utility of Rb+ and Tl+ as K+ substitutes is supported by similarities in Rb+ and Tl+ association with DNA. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-058-8_10 Inhibitors of cholesterol absorption have been sought for decades as a means to treat and prevent cardiovascular diseases associated with hypercholesterolemia. Ezetimibe is the one clear success story in this regard, and other compounds with similar efficacy continue to be sought. In the last decade, the laboratory mouse, with all its genetic power, has become the premier experimental model for discovering the mechanisms underlying cholesterol absorption and has become a critical tool for preclinical testing of potential pharmaceutical entities. This chapter briefly reviews the history of cholesterol absorption research and the various gene candidates that have come under consideration as drug targets. The most common and versatile method of measuring cholesterol absorption is described in detail along with important considerations when interpreting results, and an alternative method is also presented. In recent years, reverse cholesterol transport has become an area of intense new interest for drug discovery since this process is now considered another key to reducing cardiovascular disease risk. The ultimate measure of reverse cholesterol transport is sterol excretion and a detailed description is given for measuring neutral and acidic fecal sterols and interpreting the results. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-058-8_11 Collagen-induced arthritis (CIA) in mice is an animal model for rheumatoid arthritis (RA) and can be induced in DBA/1 and C57BL/6 mice using different protocols. The CIA model can be used to unravel mechanisms involved in the development of arthritis and is frequently used to study the effect of new therapeutics. The development of a CIA model in C57BL/6 mice recently enabled researchers to use knockout mice on this background for arthritis research. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-058-8_12 A large variety of mouse models for human skin and adnexa diseases are readily available from investigators and vendors worldwide. While the skin is an obvious organ to observe lesions and their response to therapy, actually treating and monitoring progress in mice can be challenging. This chapter provides an overview on how to use the laboratory mouse as a preclinical tool to evaluate efficacy of a new compound or test potential new uses for a compound approved for use for treating an unrelated disease. Basic approaches to handling mice, applying compounds, and quantifying effects of the treatment are presented. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-058-8_13 By definition, animal models provide only an approximation of clinical reality. One reason for this, for example, is that although metastases are the primary cause of mortality from neoplasia, by are rarely considered a target in drug discovery and development. Due to the impact of metastasis on clinical disease, we posit that metastasis should be considered in drug discovery, in addition, to more traditional biologic concepts, including drug pharmacology and toxicity. Drug discovery and developmental studies can incorporate orthotopic and spontaneous metastasis models (syngeneic and xenogeneic) with their inherent host–tumor microenvironmental interactions, in addition to confirmatory autochthonous and/or genetically engineered models (GEMs). This requires a rational and hierarchical approach using models of metastatic disease optimally using resected, orthotopic primary tumors and clinically relevant outcome parameters. In this chapter, we provide protocols for models of metastasis that can be used in translational and drug discovery studies. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-058-8_14 In testing novel anticancer therapies, researchers strive to utilize models that reflect the human disease as much as feasible. In this regard, orthotopic models are frequently developed because cancer cells in these models form tumors in, and metastasize from, a tissue environment similar to the tissue of origin of the cancer cells. Here we adapted an orthotopic colorectal cancer model, in which HT-29 colorectal cancer cells form tumors in the rectal lining and metastasize to the para-aortic lymph nodes with high frequency. Firefly luciferase-expressing HT-29 cells were used in this model to realize the benefits of bioluminescence imaging (BLI). A combination of irinotecan, 5-fluorouracil (5-FU), and leucovorin (LV) (IFL) was used as a standard chemotherapeutic regimen positive control. BLI allowed for the demonstration of the effects of IFL on tumor growth in the rectal lining, with tumor weight measurements at the end of the study reflecting total tumor burden. BLI also allowed relatively easy demonstration of reduced tissue metastasis with IFL treatment, compared to more time-consuming histological techniques. It is concluded that the orthotopic colorectal cancer model approach described represents a valuable tool for validating treatment strategies in this indication. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-058-8_15 Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by increased proliferation of granulocytic cells without the loss of their capability to differentiate. CML is derived from the hematopoietic stem cells (1) with the Philadelphia chromosome resulting from of a reciprocal translocation between the chromosomes 9 and 22 t(9;22)-(q34;q11). This translocation produces a fusion gene known as BCR-ABL which acquires uncontrolled tyrosine kinase activity, constantly turning on its downstream signaling molecules/pathways, and promoting proliferation of leukemia cell through anti-apoptosis and acquisition of additional mutations. To evaluate the role of each critical downstream signaling molecule of BCR-ABL and test therapeutic drugs in vivo, it is important to use physiological mouse disease models. In this chapter, we describe a mouse model of CML induced by BCR-ABL retrovirus (MSCV-BCR-ABL-GFP; MIG-BCR-ABL) and how to use this model in translational research. 2010-01-01T05:00:00Z