Crowding effects in Enzymatic Restriction Reactions within DNA Nanostructures

author: Matteo Castronovo, ELETTRA - Sincrotrone Trieste
published: Feb. 12, 2008,   recorded: October 2007,   views: 233

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We have studied the effect of restriction enzyme digestion reactions (DPN II) within DNA nanostructures on flat gold substrates by Atomic Force Microscopy (AFM). Typically we work with a few patches of Self Assembled Monolayers (SAMs) DNA that are hundred nm in size and are fabricated within alkylthiol SAMs on gold films by means of an AFM based lithographical technique known as Nanografting.1,2 We start by nanografting a few patches of a single stranded DNA (ssDNA) molecule of 44 base pairs (bps) with a recognition sequence of 4 bps (specific for the DPN II enzyme) in the middle. Afterwards, to obtain reaction ready DNA, the nanopatches are hybridized with a complementary ssDNA sequence of the same length. The enzymatic reactions were carried out over nanopatches with different molecular density and different geometries. Using nanopatch height measurements carried out with an AFM we are able to show that the capability of the DPN II enzyme to reach and react at the recognition site significantly depends on the geometry and the molecular density of the nanopatches. In general it was found that the digestion of the DNA by the enzyme it is strongly inhibited at a relatively low dsDNA density. Reference experiments were similarly carried out with nanopatches of a DNA sequence without the recognition site and it was found that in that case the enzymatic reaction didn’t lead to digestion of the nanopatches. These findings suggest that, due to the enzyme size, it is possible to tune the efficiency of an enzymatic reaction on a surface by changing the crowding conditions of the DNA in the nanopatches.

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