Matteo Castronovo - Control of Steric Hindrance on Restriction Enzyme Reactions with Surface-Bound DNA Nanostructures

Document created by Matteo Castronovo on Aug 22, 2014
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  Publication Details (including relevant citation   information): Castronovo, Matteo, Radovic, Slobodanka,   Grunwald, Christian, Casalis, Loredana, Morgante, Michele,   Scoles, Giacinto, 2008, 8 (12), pp   4140-4145

  Abstract: To understand better enzyme/DNA   interactions and to design innovative detectors based on DNA   nanoarrays, we need to study the effect of nanometric confinement   on the biochemical activity of the DNA molecules. We focus on the   study of the restriction enzyme reactions (Dpnll) within DNA   nanostructures on flat gold films by atomic force microscopy   (AFM). Typically we work with a few patches of DNA self assembled   monolayers (SAMs) that are hundred nm in size and are   lithographically fabricated within alkylthiol SAMs by AFM   nanografting. We start by nanografting a few patches of a   single-stranded DNA (ssDNA) molecule of 44 base pairs (bps) with   a 4 bps recognition sequence (specific for Dpnll) in the middle.   Afterwards, reaction-ready DNA nanopatches are obtained by   hybridization with a complementary 44bps ssDNA sequence. The   enzymatic reactions were carried out over nanopatches with   different density. By carrying out AFM height measurements, we   are able to show that the capability of the Dpnll enzyme to reach   and react at the recognition site is easily varied by controlling   the DNA packing in the nanostructures. We have found strong   evidence that inside our ordered DNA nanostructures the enzyme   (that works as a dimer) can operate down to the limit in which   the space between adjacent DNA molecules is equal to the size of   the DNA/enzyme complex. Similar experiments were carried out with   a DNA sequence without the recognition site, clearly finding that   in that case the enzymatic reaction did not lead to digestion of   the molecules. These findings suggest that it is possible to tune   the efficiency of an enzymatic reaction on a surface by   controlling the steric hindrance inside the DNA nanopatches   without vary any further physical or chemical variable. These   findings are opening the door to novel applications in both the   fields of biosensing and fundamental biophysics.

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