Comparison of library screening techniques used in the development of dsDNA ligands

Bioorganic and Medicinal Chemistry Letters

Volumn 13, Issue 1, 2003, Pages 47-50

  Chaltin, Patrick ^a   Borgions, Filip ^a   Van Aerschot, Arthur ^a   Herdewijn, Piet ^a  

^a REGA INSTITUTE FOR MEDICAL RESEARCH   (Belgium)

Author keywords

[No Author keywords available]

Indexed keywords

AMINO ACID DERIVATIVE; DNA; LIGAND; AMINO ACID; FLUORESCENT DYE; OLIGOPEPTIDE; PEPTIDE LIBRARY;

ARTICLE; BINDING AFFINITY; CONTROLLED STUDY; DNA BINDING; DNA LIBRARY; FLUORESCENCE; FLUORESCENT INTERCALATOR DISPLACEMENT; GEL RETARDATION; INTERMETHOD COMPARISON; PROTEIN SYNTHESIS; SCREENING TEST; TECHNIQUE; CHEMISTRY; COMPARATIVE STUDY; DRUG SCREENING; GEL MOBILITY SHIFT ASSAY; METABOLISM; METHODOLOGY; SYNTHESIS;

AMINO ACIDS; DNA; DRUG EVALUATION, PRECLINICAL; ELECTROPHORETIC MOBILITY SHIFT ASSAY; FLUORESCENT DYES; LIGANDS; OLIGOPEPTIDES; PEPTIDE LIBRARY;

EID: 0037420999     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0960-894X(02)00836-3     Document Type: Article

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16. For the ethidium bromide experiments, the same target sequence was used as originally applied in the gel shift screening experiments [5′-(AGATTGTGCAATGT)-3′:5′-(ACATTGCACAATCT)-3′]. Wells of Costar black 96-well plates were loaded with 2 μL of a 50 μM dsDNA solution, 2 μL of a 0.35 mM EtBr solution and a varying volume of oligopeptides (individual or mixtures) to obtain the necessary concentrations. The appropriate volume of a Tris/NaCl buffer (10 mM Tris-10 mM NaCl pH, 7.4) was added to obtain a total volume of 100 μL per well. Before adding the DNA to the wells, it was rendered double-stranded by placing equal amounts of the two complementary strands for 3 min at 80 °C, at room temperature for 5 min and at 4 °C for 20 min. After incubation at room temperature for 30 min, each well was read on a FL600 Microplate Fluorescence reader, with 530/25 nm as excitation wavelength and 590/35 nm as the emission detection wavelength. Two control wells (no agent=100% fluorescence, no DNA=0% fluorescence) were used per 12 samples. Fluorescence readings are reported as % fluorescence relative to the control wells. For dissociation constant determinations, several oligopeptide concentrations are used. Generally two to three sets of measurements were performed to calculate average values.