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Periodic Report Summary 1 - TROJAN-LIPID-SENSOR (Trojan-Lipid-Sensor)

The goal of my project is to develop molecular RNA tools, which I called Trojan Lipid Sensors, to enable highly localised, accurate, real-time imaging of phospholipids in living cells without the use of any cell transfection techniques. These RNA molecules bind to conditionally fluorescent molecules that are structurally related to the GFP Chromophore and are called Spinach. Spinach can be used to create small molecule sensors. If a small molecule–binding aptamer and Spinach share the critical stem required for Spinach fluorescence, small-molecule can fold the aptamer and stabilise the stem, resulting in fluorescence. Spinach can therefore function as a sensor that becomes fluorescent only upon binding to specific target molecules, in my case lipid molecules. Fusion of Spinach biosensor with Trojan Peptides will led to a new pharmacological tool to be used for phospholipid live imaging.
During the first year of outgoing phase of the project I worked on selecting the best RNA sequence that binds to Phosphatidic acid and PI(4,5)P2 using novel liposome-based RNA library selection. Secondly I worked on optimising Sensor Fluorescence by working on new RNA molecule called Corn. Corn has higher fluorescence intensity compared to Spinach and seems to be more suitable to be converted into a biosensor. For that reason I worked on making Corn as sensor using different strategies based on the high stability of specific RNA structures as 3 way junctions-based structures (3WJ).
In the second year of outgoing phase I focused on working on sensors for PA and PIP2 creating libraries with 3WJ as stabilising core structure and making circular RNA based-sensor. Moreover in order to acquire expertise on imaging membrane phospholipids, I exploited a novel sensor for Phosphatidic acid (PASS, see: Zhang F. et al. Mol Cell Biol. 2014 Jan;34(1):84-95. doi: 10.1128/MCB.00987-13). I working with eGFP-PASS biosensor helped me in finding the best conditions for transfection and imaging on Hek293 and THP-1 cells to correlate phospholipids dynamics with respect to actin polymerisation in clusters of protruding podosomes (THP-1 cells) differentiated in vitro towards immature Dendritic Cells. This has provide an additional approach for imaging the desired lipids, thus contributing to the achievement of the future experimental objectives.

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Life Sciences
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