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Content archived on 2024-06-18

Single-Molecule Fluorescence Pulsed Interleaved Excitation, a novel tool to study biomolecular interactions

Final Report Summary - SMF-PIE (Single-Molecule Fluorescence Pulsed Interleaved Excitation, a novel tool to study biomolecular interactions)

The possibility of directly detect and watch individual molecules, in real-time, and see them interacting and working is a reality available from a few years. The advances in sensitive detection devices and laser excitation sources have enabled the direct detection of single molecules and a new type of molecular spectroscopy. These new methodologies have opened different ways by which scientist interrogate systems in fields like chemistry and biology. One of these single molecule methodologies is based on the detection of the light emitted by a special type of reporter molecule called fluorophore. The detection of this light by high-performance optical microscopes and very sensitive photon detectors has given rise to single-molecule fluorescence techniques (SMF). The molecular systems can be directly observed, one by one, by attaching fluorophores to the relevant studied species, allowing detection, localisation, and the study of interactions within the relevant system of interest. A very powerful approach is the use of two different fluorophores emitting light of different colour, in separate regions of the visible electromagnetic spectrum. Attaching two different fluorophores to different sections of the same biomolecule allows to study conformational changes within functional activity, for instance, to follow protein and RNA folding or conformational changes occurring within enzymatic activity. Another alternative is attaching different colour fluorophores to separate molecules. With this scheme, one can probe direct interactions between biomolecules, extract the stoichiometry of interaction of different subparts of a biomolecular complex, or follow aberrant aggregation of proteins, related to one of the current major biomedical problems, such as Alzheimer's and Parkinson's diseases. Against this background, the three broad aspects of current research in the SMF field are:
1) technical developments of the SMF methodologies;
2) development of improved fluorophores to enhance the quality of the SMF results, and
3) application of the SMF experiments to relevant biological and biomedical problems, to take advantage of the interesting capabilities of detecting individual molecules one by one.

Dr Angel Orte was appointed as the researcher to hold the European Reintegration Grant, at the University of Granada (Spain), after having held a Marie Curie Intra-European Fellowship at the University of Cambridge (U. K.). The main goal of the grant was to provide Dr Orte with resources to start independent research, transfer the knowledge acquired during his post-doctoral experience to the host country, helping the fellow to reach a mature position in Academia.

The first task that Dr Orte tackled was to transfer his knowledge on SMF techniques, acquired during the IEF period, to the host group at the University of Granada (UGR). This knowledge on SMF techniques has resulted in the publication of two review articles on the applications of dual-colour SMF and single molecule FRET to study biological systems. Dr Orte implemented within the host group one of the most advance dual-colour SMF techniques, the so-called Pulsed Interleaved Excitation (PIE). Likewise, he provided appropriate training to the other members of the research group.

Once the technique was in place, one of the most successful objectives of the project concerned the development of improved fluorophores to enhance the quality of the SMF results. The chemical modification of fluorescent molecules, called xanthenes, at specific positions showed improved performance for SMF techniques. One of these xanthene derivative has shown to be an excellent reporter of the velocity of proton transfer reactions in aqueous media, especially when this transfer is facilitated by other molecules such as phosphate ions. This sets up the basis for the development of a potential intracellular phosphate sensor. The full characterisation of the behaviour of these xanthenes derivatives in aqueous solution, and in media mimicking the intracellular environment (crowding agents or micelles) has resulted in the publication of six different papers, and many communications in international conferences, as detailed below.

The following objective was to employ SMF techniques to study a special kind of fluorescent molecules, the cyanine fluorophores, which have the feature of undergo an enhancement of their fluorescence emission upon intercalation into the double-stranded DNA. This is why these fluorophores are known as DNA intercalators. This family of fluorophores are currently broadly used for DNA staining and emerging superresolution microscopy to image chromosomes. The interaction behaviour of one of the cyanine fluorophores, called BOBO-3, with double-stranded DNA in concentrated solutions and at the single molecule level has been thoroughly described in three papers published in international journals. The most striking results were the characterisation of different binding modes of the fluorescent intercalator with the DNA, including a very specific interaction between cytosine bases and an intra-molecular structural rearrangement of BOBO-3, called H-aggregate. This latter interaction mode is so tight that when it is formed, it even hinders the hybridisation of complementary strands of the DNA. All these results have established a theoretical basis that is being applied to other molecules of the cyanine family.

The next objective involved the characterisation of the early stages of the amyloid fibril formation reaction, the aberrant aggregation of proteins that occurs in diseases like Alzheimer's and Parkinson's, using SMF techniques. The misfolded aggregation of proteins is a major biomedical concern, and the understanding of the mechanisms of amyloid formation and cytotoxicity has become a major challenging the biophysics field. This objective was the most ambitious and challenging of the original work plan. This study needed a set of preliminary milestones involving the appropriate design of the model protein, and optimisation of the conditions to attach a reporter fluorophore on it. Reaching these milestones turned out to be a challenging and time-consuming task. By the end of the project, in December 2011, all the initial issues were overcome and everything was in place to start the planned experiments, that could not be finished due to the limited time of the ERG grant and the broad and different research lines tackled over the fellowship period. Nevertheless, the use of a minimal amount of labelled protein in the presence of unlabelled protein has provided interesting preliminary results that show that amyloid fibrils are formed with the fluorophore, and that the fluorophore does not alter the main kinetic of amyloid formation. This research line has many interesting future prospects, since Dr Orte, as PI, has been awarded a research grant for 4 years to continue with such line.

Apart from the results related to the main research project, the ERG grant has resulted in a valuable tool to establish long-lasting collaborations with Prof Klenerman's group at the University of Cambridge (U. K.), Prof Elisabeth Hall's group at the Univerisity of Cambridge (U. K.), and Prof Boens'group at the K. U. Leuven (Belgium). Dr Orte has fruitfully continued collaborating with Prof Klenerman's group in SMF studies of protein unfolding and function. This collaboration has resulted in four publications and two more manuscripts currently under review. The collaboration with Prof Hall in the development of lifetime-based Quantum Dots fluorescent nanosensors has resulted in two publications in international journals so far. Likewise, the collaboration with Prof Boens'group in the development of novel BODIPY dyes has resulted in two manuscripts that will be submitted soon.

Finally, the ERG grant has meant an impact in the career of the fellow. Dr Orte has been successfully integrated within the FQM-247 research group, having held a postdoctoral position within the funded project CTQ2007-61619 from the Spanish Ministry of Education and Science until December 2009. In December 2009, Dr Orte has been awarded an Assistant Lecturer position, a 5-year duration lectureship. This position has prospects of stabilisation at the end of the contract, should the Department provide positive evaluation. Obtaining the ERG has provided Dr Orte with certain research independency and renowned merits in his curriculum vitae that definitively has helped him towards a mature permanent position in Academia.