• Home
  • Search Results
  • Quantitative detection of small molecule/DNA complexes employing a force-based and label-free DNA-microarray.

Quantitative detection of small molecule/DNA complexes employing a force-based and label-free DNA-microarray.

Biophysical journal (2009-06-03)
Dominik Ho, Christian Dose, Christian H Albrecht, Philip Severin, Katja Falter, Peter B Dervan, Hermann E Gaub
ABSTRACT

Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA.ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA.ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA.ligand complexes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, fatty acid free, essentially globulin free, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, essentially globulin free, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, fatty acid free, low endotoxin, lyophilized powder, BioReagent, suitable for cell culture, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, cold ethanol fraction, pH 5.2, ≥96%
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially fatty acid free, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, suitable for RIA, pH 5.2, ≥96%
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 5.2, ≥96%
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, lyophilized powder, essentially fatty acid free, ≥98% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, suitable for (for molecular biology), Non-acetylated
Sigma-Aldrich
Bovine Serum Albumin, pH <5.0, powder
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, crystallized, ≥98.0% (GE)
Sigma-Aldrich
Bovine Serum Albumin, powder, BioXtra
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, suitable for hybridization, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially globulin free, ≥99% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially IgG-free, low endotoxin, BioReagent, suitable for cell culture
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially fatty acid free and essentially globulin free, ≥99% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, low endotoxin, lyophilized powder, Cohn Analog, BioReagent, suitable for cell culture, ≥98% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, Cohn Fraction V, lyophilized powder, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 5.2, ≥96%
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, low endotoxin, suitable for cell culture, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, chromatographically purified, New Zealand origin, low endotoxin, suitable for cell culture, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially globulin free, BioReagent, suitable for cell culture
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially globulin free, low endotoxin, ≥98% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, ≥98% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, 1× crystallized, ≥97% (agarose gel electrophoresis)