Computer-Aided Design of DNA-Based Nanoinstruments
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The formation of protein–DNA complexes is a crucial step in many biological processes. Structural information on such complexes originates mainly from X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. However, only few experimental techniques are available to deduce structural information on larger flexible protein–DNA complexes. We have designed DNA-based nanoinstruments that enable us to measure DNA deformation in solution.
Using bioinformatics and conformational search force field methods, we designed DNA molecules with one, two, and three dA5 bulges separated by double-stranded sections of DNA to be used as substrates for DNA–protein binding assays. The molecules were synthesized and 5′-end-labeled with 6-fluorescein (Fl) and 5-tetramethylrhodamine (TMRh) dyes. These large DNA molecules adopt a nearly planar conformation in aqueous solution with their helix ends being close in space. Fluorescence resonance energy transfer (FRET) between dyes was used to measure end-to-end distances and monitor the binding of proteins such as catabolite activator protein (CAP). The changes in the FRET efficiency of three-bulge DNA structures containing the specific binding sequence of CAP demonstrated significant deformation of the DNA upon binding of CAP.
In addition, the NMR solution structure of the dA5 bulge DNA fragment was solved.2 This structure together with explicit positions of the dyes at the DNA helix ends allowed us to translate the measured FRET data into precise structural information.3 This was demonstrated with two further DNA-binding proteins, high-mobility-group protein (HMG-D) and integration host factor (IHF). The concept of DNA-based nanoinstruments detecting DNA deformations can be used in solution, in gels, and at surfaces. It allows one to measure the influence of the DNA sequence itself, of proteins, and of environmental factors.