Abstract
Single-molecule methods have become crucial biophysical tools for investigating DNA-protein interactions in vitro. A general drawback with most single DNA molecule techniques is that the DNA must be tethered in at least one of its end. This means, for example that reactions involving two DNA ends are tricky to study. Nanofluidic tools have frequently been used in static DNA studies, where the molecule is stretched to an extension close to its contour length without any tethering. However, studying dynamic reactions in real-time is a large challenge since it is not possible to actively control the environment within the nanofluidic channel, without holding on to the molecule. We have developed a nanofluidic device that can be used to study dynamic processes on single DNA molecules in real-time. This novel design enables active control of the local environment in the nanofluidic channel, while keeping the DNA molecule confined. Using this device, we are able to add analytes, such as DNA-binding proteins, on demand and simultaneously study the response of the DNA molecule by fluorescence imaging. We are thus able to map the interplay between biomolecules in order to reveal the unknown details of different biological processes. As a proof of concept, DNase I was successfully added to nanoconfined DNA stained with YOYO-1 in order to induce digestion on demand. To further show the functionality of the nanofluidic device, we have studied real-time interactions between DNA and proteins known to change the physical properties of DNA upon binding. The results clearly demonstrate that we are able to control the reaction conditions inside the nanofluidic channels, and thereby study complex biomolecular processes in vitro.
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