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Methods (San Diego, Calif.) Feb 1997
Review
Topics: Animals; DNA; DNA Footprinting; DNA-Binding Proteins; Polymerase Chain Reaction; RNA; Regulatory Sequences, Nucleic Acid
PubMed: 8993026
DOI: 10.1006/meth.1996.0400 -
Methods in Molecular Biology (Clifton,... 1997
Review
Topics: Base Sequence; DNA; DNA Footprinting; Deoxyribonuclease I; Humans; Molecular Sequence Data
PubMed: 9407524
DOI: 10.1385/0-89603-447-X:1 -
Nature Communications Aug 2017Most DNA processes are governed by molecular interactions that take place in a sequence-specific manner. Determining the sequence selectivity of DNA ligands is still a...
Most DNA processes are governed by molecular interactions that take place in a sequence-specific manner. Determining the sequence selectivity of DNA ligands is still a challenge, particularly for small drugs where labeling or sequencing methods do not perform well. Here, we present a fast and accurate method based on parallelized single molecule magnetic tweezers to detect the sequence selectivity and characterize the thermodynamics and kinetics of binding in a single assay. Mechanical manipulation of DNA hairpins with an engineered sequence is used to detect ligand binding as blocking events during DNA unzipping, allowing determination of ligand selectivity both for small drugs and large proteins with nearly base-pair resolution in an unbiased fashion. The assay allows investigation of subtle details such as the effect of flanking sequences or binding cooperativity. Unzipping assays on hairpin substrates with an optimized flat free energy landscape containing all binding motifs allows determination of the ligand mechanical footprint, recognition site, and binding orientation.Mapping the sequence specificity of DNA ligands remains a challenge, particularly for small drugs. Here the authors develop a parallelized single molecule magnetic tweezers approach using engineered DNA hairpins that can detect sequence selectivity, thermodynamics and kinetics of binding for small drugs and large proteins.
Topics: Base Sequence; Binding Sites; DNA; DNA Footprinting; Kinetics; Ligands; Magnetics; Models, Genetic; Nucleic Acid Conformation; Optical Tweezers; Thermodynamics
PubMed: 28824174
DOI: 10.1038/s41467-017-00379-w -
Methods in Molecular Biology (Clifton,... 2021The in vivo footprinting method identifies protein-targeted DNA regions under different conditions such as carbon sources. Dimethyl sulfate (DMS) generates methylated...
The in vivo footprinting method identifies protein-targeted DNA regions under different conditions such as carbon sources. Dimethyl sulfate (DMS) generates methylated purine bases at DNA sites which are not bound by proteins or transcription factors. The DNA is cleaved by HCl, and the resulting DNA fragments are 5'-end [6-FAM]-labeled by a linker-mediated PCR (LM-PCR). Fluorescent fragments are separated and analyzed on a capillary sequencer, followed by automated data analysis using the software tool ivFAST.
Topics: Base Sequence; DNA Footprinting; DNA, Fungal; Electrophoresis, Capillary; Hypocreales; Methylation; Polymerase Chain Reaction; Promoter Regions, Genetic
PubMed: 33165789
DOI: 10.1007/978-1-0716-1048-0_15 -
Methods in Molecular Biology (Clifton,... 2009Various methodologies have been developed for the detection of DNA-binding activities and the identification of the "footprints" of a protein on DNA. The most widely...
Various methodologies have been developed for the detection of DNA-binding activities and the identification of the "footprints" of a protein on DNA. The most widely used footprinting techniques employ reagents such as deoxyribonuclease I (DNase I) and dimethyl sulfate (DMS) for protection analysis in solution. Nevertheless, these techniques have several disadvantages, and although these may be bypassed by coupling the footprinting reaction with an electrophoretic mobility-shift assay (EMSA), the size and the sequence specificity of DNase I and DMS as well as the problem of protein exchange during the footprinting reaction pose significant limitations. These limitations can be circumvented by combining the advantages of EMSA, with the subsequent exposure of the resolved DNA-protein complex(es) to the chemical nuclease 1,10-phenanthroline-copper ion (OP-Cu) while they are still embedded in the polyacrylamide matrix (in-gel assay).
Topics: Animals; Copper; DNA; DNA Footprinting; Electrophoresis, Polyacrylamide Gel; Electrophoretic Mobility Shift Assay; Kinetics; Phenanthrolines; Protein Binding; Proteins; Sequence Analysis, DNA
PubMed: 19378167
DOI: 10.1007/978-1-60327-015-1_13 -
Methods in Molecular Biology (Clifton,... 2007Phylogenetic footprinting is powerful technique for finding functional elements from sequence data. Functional elements are thought to have greater sequence constraint...
Phylogenetic footprinting is powerful technique for finding functional elements from sequence data. Functional elements are thought to have greater sequence constraint than nonfunctional elements, and, thus, undergo a slower rate of sequence change through time. Phylogenetic footprinting uses comparisons of homologous sequences from closely related organisms to identify "phylogenetic footprints," regions with slower rates of sequence change than background. This does not require prior characterization of the sequence in question, therefore, it can be used in a wide range of applications. In particular, it is useful for the identification of functional elements in noncoding DNA, which are traditionally difficult to detect. Here, we describe in detail how to perform a simple yet powerful phylogenetic footprinting analysis. As an example, we use ribosomal DNA repeat sequences from various Saccharomyces yeasts to find functional noncoding DNA elements in the intergenic spacer, and explain critical considerations in performing phylogenetic footprinting analyses, including the number of species and species range, and some of the available software. Our methods are broadly applicable and should appeal to molecular biologists with little experience in bioinformatics.
Topics: Base Sequence; DNA Footprinting; DNA, Fungal; DNA, Ribosomal; Molecular Sequence Data; Nucleic Acid Conformation; Phylogeny; Repetitive Sequences, Nucleic Acid; Saccharomyces cerevisiae; Sequence Homology, Nucleic Acid
PubMed: 17993686
DOI: 10.1007/978-1-59745-514-5_23 -
Cold Spring Harbor Protocols May 2013DNase I footprinting has found a wide following for both identifying and characterizing DNA-protein interactions, particularly because of its simplicity. The concept is...
DNase I footprinting has found a wide following for both identifying and characterizing DNA-protein interactions, particularly because of its simplicity. The concept is that a partial digestion by DNase I of a uniquely (32)P-end-labeled fragment will generate a ladder of fragments, whose mobilities on a denaturing acrylamide gel and whose positions in a subsequent autoradiograph will represent the distance from the end label to the points of cleavage. Bound protein prevents binding of DNase I in and around its binding site and thus generates a "footprint" in the cleavage ladder. The distance from the end label to the edges of the footprint represents the position of the protein-binding site on the DNA fragment. The position of the binding site can be determined by electrophoresing a DNA sequencing ladder alongside the footprint. DNase I cannot bind directly adjacent to a DNA-bound protein because of steric hindrance. Hence, the footprint gives a broad indication of the binding site, generally 8-10 base pairs (bp) larger than the site itself.
Topics: Binding Sites; DNA; DNA Footprinting; DNA-Binding Proteins; Deoxyribonuclease I; Isotope Labeling; Phosphorus Radioisotopes
PubMed: 23637368
DOI: 10.1101/pdb.prot074328 -
Biochemical Society Transactions Aug 2008Transcription is often regulated at the level of initiation by the presence of transcription factors or nucleoid proteins or by changing concentrations of metabolites....
Transcription is often regulated at the level of initiation by the presence of transcription factors or nucleoid proteins or by changing concentrations of metabolites. These can influence the kinetic properties and/or structures of the intermediate RNA polymerase-DNA complexes in the pathway. Time-resolved footprinting techniques combine the high temporal resolution of a stopped-flow apparatus with the specific structural information obtained by the probing agent. Combined with a careful quantitative analysis of the evolution of the signals, this approach allows for the identification and kinetic and structural characterization of the intermediates in the pathway of DNA sequence recognition by a protein, such as a transcription factor or RNA polymerase. The combination of different probing agents is especially powerful in revealing different aspects of the conformational changes taking place at the protein-DNA interface. For example, hydroxyl radical footprinting, owing to their small size, provides a map of the solvent-accessible surface of the DNA backbone at a single nucleotide resolution; modification of the bases using potassium permanganate can reveal the accessibility of the bases when the double helix is distorted or melted; cross-linking experiments report on the formation of specific amino acid-DNA contacts, and DNase I footprinting results in a strong signal-to-noise ratio from DNA protection at the binding site and hypersensitivity at curved or kinked DNA sites. Recent developments in protein footprinting allow for the direct characterization of conformational changes of the proteins in the complex.
Topics: DNA; DNA Footprinting; Kinetics; Protein Binding; Proteins; Time Factors
PubMed: 18631151
DOI: 10.1042/BST0360745 -
Methods in Enzymology 2001RNase I and RNase T1 can be used to obtain high-quality footprinting information for paromomycin binding to a 176-mer RNA from the packaging region of HIV-1 (LAI)....
RNase I and RNase T1 can be used to obtain high-quality footprinting information for paromomycin binding to a 176-mer RNA from the packaging region of HIV-1 (LAI). Controls and scanning procedures are necessary for quantitation of autoradiographic data, so that footprinting plots showing cutting behavior as a function of drug concentration can be used to identify binding sites and regions of altered structure on the 176-mer. From the RNase I footprinting results the primary paromomycin binding sites on the 176-mer are on the main stem and on the stem of SL1, but noncontiguous sequences may be involved in the same binding event. Strong enhancements in cleavage with added drug are also observed, indicating drug-induced structural changes. Drug binding may cause linker regions between stem-loops of the 176-mer to change structure, possibly providing a site or sites for additional drug binding. Because drug binding changes the structure of the packaging region, which may alter its function, paromomycin analogs with enhanced specificity for HIV psi RNA have potential as a new class of agent for treating AIDS.
Topics: Base Sequence; DNA Footprinting; DNA Primers; HIV-1; Ligands; Molecular Sequence Data; Nucleic Acid Conformation; Paromomycin; Pharmaceutical Preparations; RNA; RNA, Viral
PubMed: 11494862
DOI: 10.1016/s0076-6879(01)40435-6 -
Methods in Cell Biology 1995
Review
Topics: Alcohol Dehydrogenase; DNA Footprinting; DNA, Plant; DNA-Binding Proteins; Mutagens; Sulfuric Acid Esters
PubMed: 8531771
DOI: No ID Found