Super-resolution fluorescence microscopy of isolated and cell DNA

限制性内切酶是一种切割双链DNA的酶。分子生物学和基因工程的许多过程都依赖于限制性内切酶，因此它们的实际应用非常广泛。众所周知，作为其作用机制的一部分，这些酶中的一些能够拉动(转移)DNA以找到正确的切割位置，使它们成为重要的分子马达。还观察到这些酶与DNA形成环。然而，它们的确切作用机制仍然不清楚，而且已经有了不同的方案。研究限制性内切酶对DNA的易位和环化作用将使我们对这些酶有更详细的了解，从而使我们能够改进它们的性质，使其具有一定的应用。我们建议使用单分子荧光显微镜来研究DNA-酶的相互作用。单分子技术的优势在于，它们可以提供行为分布的信息，而不是平均值。此外，他们能够识别可能被总体平均所掩盖的中间物种，并允许观察罕见的事件。单分子光谱学在酶的研究中的结果已经通过揭示记忆效应等性质改变了我们看待酶的方式，并促使科学界重新解释酶催化的基本规律。这个项目将使用几种互补的单分子技术来了解限制酶机制的不同方面：-所谓DNA幕的组装，即DNA链的有序阵列，将有助于在单分子水平上高通量收集信息，并在不同的缓冲条件下筛选不同的DNA-酶对。它还将允许比较用荧光标记标记DNA和酶的不同策略。-一旦我们找到合适的DNA-酶对，我们就可以使用这个系统来尝试一种新的单分子技术，称为光活化-定位显微镜(Palm)。这项技术的目的是以低于光的衍射极限(超分辨率)的空间分辨率对样品成像。-我们将用福斯特共振能量转移(FRET)研究来补充上述研究。FRET可以作为一种方便的光谱标尺来获得1-10 nm范围内的几何信息。因此，我们可以间接获得DNA-蛋白质相互作用的信息以及相互作用时发生的构象变化。我们将使用单分子技术和本体技术来研究FRET。FRET可以通过荧光寿命的变化来监测，我们将使用荧光寿命成像显微镜(FLIM)在整体水平上监测FRET。

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.