Photothermal imaging of biomimetic nanoparticles to investigate the real-time dynamics of transcription at the single molecule level in living cells

生物学中最大的挑战之一是能够测量在单细胞中发生的生物反应。其中一个重要的过程是转录，即基因的DNA序列被聚合酶读取以产生RNA，然后RNA被翻译以产生蛋白质的反应。通过对细胞中萤火虫荧光素酶报告蛋白的合成进行成像，在单细胞中间接监测转录，因为它使活细胞发光，因此可以使用计数光子的相机进行测量。活细胞中的生物过程也可以通过显微镜使用天然荧光蛋白来监测，所述天然荧光蛋白在细胞中遗传合成并与感兴趣的蛋白融合。在过去的10年里，这导致了细胞生物学的一场革命，因为第一次可以观察单个生物过程，如蛋白质运动，因为它们发生在单细胞中。位于利物浦的细胞成像中心一直是这些技术开发和应用的领先中心。使用这些方法，通常不可能观察细胞中单个分子的运动。这将使人们对许多生物过程的理解达到一个新的水平。在单个细胞中研究转录何时被激活蛋白质（称为转录因子）开启的能力是一个重要的目标。越来越清楚的是，这些事件可能往往是由概率和平均这些过程或间接测量他们错过了重要的信息。我率先开发了仿生金纳米粒子，它可以很容易地与蛋白质结合。由于纳米颗粒的小尺寸，所得分子可以以与正常蛋白质相同的方式表现。我将建造世界上第二台光热显微镜，这将是第一台专门为研究活细胞而设计的显微镜（与波尔多的卢尼斯和利物浦的白色一起）。这种显微镜允许在光学复杂的环境中容易地观察金和银纳米颗粒。这在纳米生物技术的重要新兴领域有许多应用。使用这种新的显微镜，我们将能够长时间观察活细胞内的单个纳米颗粒，而不会丢失任何信号，也不会损坏细胞。我将继续开发仿生纳米颗粒，并优化它们与功能蛋白质和其他分子偶联的能力，这些分子可用于标记生物过程（与Brust，利物浦和Desbat，波尔多合作）。目前，有必要将蛋白质结合到细胞外试管中的纳米颗粒上，然后将所得缀合物引入细胞中。这具有的缺点是纯化蛋白质需要时间，并且具有蛋白质可能不具有完全功能的风险。因此，我将开发一种新的方法，将纳米颗粒结合到细胞内的蛋白质上（与Johnsson，洛桑合作）。纳米颗粒将被引入细胞中，在那里它们可以特异性地与感兴趣的蛋白质偶联。我将利用这项技术来研究活细胞中单个基因的转录。我将使用金和银纳米粒子的组合，它们可以相互区分，以便同时观察不同的过程。第一个目标将是使用三螺旋形成寡核苷酸（与特定的双链DNA靶序列形成稳定和特异性的相互作用）来鉴定哺乳动物细胞核中单个基因的位置（与杰克逊，曼彻斯特和乔瓦尼angeli，巴黎）。然后，我将使用纳米粒子来研究单个转录因子分子与基因的结合。我将通过在RNA中引入蛋白质结合位点来标记基因，这样通过转录产生的早期RNA也可以被可视化。这将首次允许在单个基因上研究基因启动的过程。

One of the greatest challenges in biology is to be able to measure biological reactions as they happen in single cells. One important process is transcription, the reaction in which the DNA sequence of a gene is read by a polymerase to produce an RNA which is then translated to produce a protein. Transcription has been monitored indirectly in single cells through imaging of the synthesis of the firefly luciferase reporter protein in cells since it makes living cells glow and can therefore be measured using a camera that counts photons. Biological processes in living cells can also be monitored by microscopy using naturally fluorescent proteins which are genetically synthesised in the cell fused to proteins of interest. Over the past 10 years this has led to a revolution in cell biology because for the first time single biological processes such as protein movement can be watched as they happen in single cells. The Centre for Cell Imaging in Liverpool has been a leading centre in the development and application of these technologies. Using these approaches it is not generally possible to watch the movement of single molecules in cells. This would allow a new level of understanding of many biological processes. The ability to study in a single cell exactly when transcription is switched on by activating proteins (called transcription factors) is an important objective. It is becoming clear that these events may often be governed by probability and that averaging such processes or measuring them indirectly misses important information. I pioneered the development of biomimetic gold nanoparticles which can be easily coupled to proteins. Due to the small size of the nanoparticles the resulting molecules can behave in the same way as the normal protein. I will build the world's second photothermal microscope, which will be the first to be specifically designed to study living cells (with Lounis, Bordeaux, and White, Liverpool). This microscope allows the easy visualisation of gold and silver nanoparticles in optically complex environments. This has many applications in the important emerging field of nanobiotechnology. Using this new microscope we will be able to see single nanoparticles within living cells for long periods of time without any loss of signal and without damaging the cell. I will continue to develop biomimetic nanoparticles and optimise their ability to couple to functional proteins and other molecules that can be used to label biological processes (with Brust, Liverpool, and Desbat, Bordeaux). At present it would be necessary to bind proteins to the nanoparticles in the test tube outside the cell and then to introduce the resulting conjugate into the cell. This has the disadvantage that it takes time to purify the protein and has the risk that the protein may not be fully functional. I will therefore develop a new methodology for binding nanoparticles to proteins within cells (with Johnsson, Lausanne). The nanoparticles will be introduced into the cell where they can specifically couple with the protein of interest. I will use this technology to study transcription at single genes in living cells. I will use a combination of gold and silver nanoparticles which can be distinguished from each other to allow different processes to be watched at the same time. The first aim will be to use triple helix forming oligos (which form stable and specific interactions with specific double stranded DNA target sequences) to identify the position of single genes in the mammalian cell nucleus (with Jackson, Manchester, and Giovannangeli, Paris). I will then use nanoparticles to study the binding of single transcription factor molecules to the gene. I will mark the gene by introducing protein binding sites into the RNA so that the early RNA produced by transcription can be also be visualised. This will for the first time allow the processes that switch genes on to be studied at single genes.