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

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.

The concept of signalling pathways is being replaced by a new paradigm of regulatory networks. The understanding of these networks doesn't require only the knowledge of the interactions between the elements but also the knowledge of the dynamics of these interactions on different time scales. Some crucial elements are present at low copy numbers in a single cell and current approaches based on fluorescence imaging lack single molecule sensitivity. Advances in nanosciences offer new tools to increase biological understanding. I propose to combine two recent breakthroughs in the fields of nanoparticle detection and nanoparticle preparation to effectively reach the goal of real-time single molecule imaging in living cells. The first breakthrough was the development of single gold nanoparticle detection down to 1.8 nm by photothermal heterodyne microscopy [Boyer et al., Science, 2002]. The second breakthrough was the preparation of highly versatile protein-like nanoparticles using peptides that spontaneously form a self-assembled monolayer on gold [Levy et al., J. Am. Chem. Soc., 2004]. The peptide monolayer can be tailored to obtain a variety of bioconjugates. The research programme proposes: 1) To build a photothermal microscope specially designed for long-term imaging of single nanoparticles in living cells; 2) To develop gold and silver nanoparticle probes that will target transcription factors, mRNA and specific genes within the nucleus; 3) To visualize these probes in real time to explore the dynamics of the transcription regulatory network. The combined analysis of transcription factor binding and transcription initiation at the single molecule level will give new insights into the dynamic and stochastic processes that regulate transcription at single genes in single cells. It will also provide valuable information and tools that are relevant for gene therapy, drug design and drug delivery.