Super-resolution fluorescence microscopy of isolated and cell DNA

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A restriction enzyme is an enzyme that cuts double-stranded DNA. Many of the procedures of molecular biology and genetic engineering rely on restriction enzymes, and thus their practical application is widespread. It is known that as part of their mechanism of action, some of these enzymes are able to pull ( translocate ) DNA in order to find the right place to cut, making them important molecular motors . It has also been observed that the enzymes form loops with DNA. However, their exact mechanism of action is still unknown, and different schemes have been postulated. The study of the translocation and looping of DNA by restriction enzymes will allow us to gain a detailed understanding of these enzymes and, in turn, will allow us to improve their properties for a certain application.We propose the use of single-molecule fluorescence microscopy to study the DNA-enzyme interaction. The advantages of single-molecule techniques are that they can provide information of a distribution of behaviours, rather than an average value. Moreover, they are able to identify intermediate species that would be obscured by ensemble averaging, and allow the observation of rare events. The results of single-molecule spectroscopy in the study of enzymes have already changed the way we look at them by uncovering properties such as memory effects, and have prompted the scientific community to reinterpret fundamental laws of enzyme catalysis.Several complementary single-molecule techniques will be used in this project to understand different aspects of the mechanism of restriction enzymes:- The assembly of the so-called DNA curtains , which are organized arrays of DNA strands, will be useful to gather information at the single-molecule level with high throughput, and screen different DNA-enzyme pairs in different buffer conditions. It will also allow to compare different strategies to label with fluorescent tags the DNA and the enzyme. - Once we find a suitable DNA-enzyme pair, we can use this system to try a new single-molecule technique called photoactivation-localization microscopy (PALM). The purpose of this technique is to image a sample with spatial resolution below the diffraction limit of light ( super-resolution ). - We will complement the above studies with the study of Foerster resonance energy transfer (FRET). FRET can be used as a convenient spectroscopic ruler to obtain geometric information in the 1-10 nm scale. Thus, we can indirectly obtain information about DNA-protein interaction and the conformational changes occurring upon the interaction. We will use both single-molecule and bulk techniques to study FRET. FRET can be monitored through changes in fluorescence lifetime, and we will use fluorescence lifetime imaging microscopy (FLIM) to monitor FRET at the bulk level.