Periodic Reporting for period 1 - PLIM-G4 (Long-lived optical probes to image G-quadruplex DNA in live cells)
Periodo di rendicontazione: 2021-02-01 al 2023-01-31
Genomic DNA typically exists as a double helix stabilized by hydrogen bonding interactions between Watson-Crick base pairs. However, it is also known that DNA can adopt various other non-canonical conformations including hairpin, triplex, homo-duplex, quadruplex etc. depending on the DNA sequence and the intracellular environment. It has been hypothesised, and to an extent shown, that some of these non-canonical DNA structures are involved in various biological functions. Amongst these structures, four stranded structures known as G-quadruplexes (G4s) have received particular attention over the past two decades. However, to date, we are still lacking tools that allows us to visualizing G-quadruplexes directly in live cells. While several small-molecule probes that fluoresce upon interaction with DNA have been reported, none of them have been successful at imaging G-quadruplexes in a cellular environment. This is mainly due to the fact that they rely on changes in intensity which are not possible to track properly in a cellular environment. Recently, the host lab reported a novel strategy to image G-quadruplexes in live cells. This approach makes use of the changes in emission lifetime (rather than intensity) of optical probes upon their interaction with different topologies of DNA. Since lifetime is concentration independent, this approach can be successfully used to image G-quadruplexes in live cells. While this has proven to be a highly successful approach, it is still in its infancy since the probe developed so far has a number of limitations such as low brightness, relatively small lifetime range and low selectivity. Thus, this project was aimed to develop a new set of probes that could address all these issues and use them to image the dynamics of G-quadruplexes in live cells in real time via phosphorescence lifetime imaging microscopy (PLIM).
• Objective 1 (O1): Develop a high-throughput screening methodology to synthesise new ligands and Pt complexes with a range of substituents to tune their DNA selectivity (WP1).
• Objective 2 (O2): Develop automated processes to screen the phosphorescence of the new Pt-complexes (libraries from O1) in the absence and presence of different DNA structures (including G4s) (WP2).
• Objective 3 (O3): From O2, select the probes with the highest switch-on emission effect and selectivity for G4 DNA, and conduct detailed photophysical studies including the determination of affinity constants and the emission lifetime of the probes in the presence of different DNA structures (WP2).
• Objective 4 (O4): Select the probe(s) with the largest emission lifetime difference when bound to different DNA structures and study their cellular uptake and localisation using confocal microscopy (WP3).
• Objective 5 (O5): Following O4, conduct Phosphorescence Lifetime Imaging Phosphorescence (PLIM) with the selected probe(s) on live cells to establish the distribution of G4s. Different eukaryotic cell types (e.g. cancer vs. non-cancer) and at different stages of the cell cycle will be studied (WP4).
• Objective 1 (O1): Develop a high-throughput screening methodology to synthesise new ligands and Pt complexes with a range of substituents to tune their DNA selectivity (WP1).
• Objective 2 (O2): Develop automated processes to screen the phosphorescence of the new Pt-complexes (libraries from O1) in the absence and presence of different DNA structures (including G4s) (WP2).
• Objective 3 (O3): From O2, select the probes with the highest switch-on emission effect and selectivity for G4 DNA, and conduct detailed photophysical studies including the determination of affinity constants and the emission lifetime of the probes in the presence of different DNA structures (WP2).
• Objective 4 (O4): Select the probe(s) with the largest emission lifetime difference when bound to different DNA structures and study their cellular uptake and localisation using confocal microscopy (WP3).
• Objective 5 (O5): Following O4, conduct Phosphorescence Lifetime Imaging Phosphorescence (PLIM) with the selected probe(s) on live cells to establish the distribution of G4s. Different eukaryotic cell types (e.g. cancer vs. non-cancer) and at different stages of the cell cycle will be studied (WP4).
This report mainly focuses on the synthesis and characterization of several tetradentate cyclometalated Pt-complexes followed by biophysical and cellular studies of the new optical probes. As part of this project, new cyclometallated platinum complexes with planar structures and helical structure were successfully synthesised. Initial in vitro DNA studies showed only one probe (L2-PtII) among the tested ones have i) reasonably selectivity for G4 DNA over other topologies; ii) very weak emission when free in solution but is highly emissive when bound to DNA; iii) displayed pronounced lifetime differences of around 1.5 µs when it is bound to duplex vs. G4 DNA. Further, we have also used the increased fluorescence intensity and lifetime of probes upon DNA binding to enable cellular imaging studies. The microscopy experiments with live U2OS cells have shown that while the newly synthesized optical probes are not cell permeable upon fixation they localize in nuclei and nucleoli. PLIM results directly confirm the ability of one of the probes to stain G4 quadruplexes within fixed cells. Therefore, for the first time, we demonstrated the possibility to visualize the G4 quadruplexes in cells by phosphorescence.
The research outcomes from the MSCA will have potential impact on DNA G4 research. The scientific impacts of the project were aimed at developing a new set of probes that address
limitations of previous optical probes such as low brightness, relatively small lifetime range and low selectivity and provide pronounced lifetime differences when it is bound to duplex vs. G4 DNA and led us to image the dynamics of G-quadruplexes in live cells in real time. Although I have developed several cyclometalated Pt-optical probes only one probe has provided PLIM results directly and confirm the ability of the probes to stain G4 quadruplexes within fixed cells. Based on the outcomes of this project it has shown that the potential of cyclometalated Pt-complexes to target G4 DNA structures. Further, this data provides basis for future development of new cyclometalated Pt-complexes with improved cells uptake and ability to visualize G4 DNA in live cells using PLIM.
The other impacts of the projects involve improving the future career prospects of the researcher after the fellowship and effective dissemination of the results obtained in this project. This fellowship has provided me a great opportunity to study and explore the fascinating and emerging research area and make the first steps towards establishing myself as an expert in interdisciplinary research filed. Further, it also helped me in broadening my scientific expertise, strengthen my skill and knowledge in chemical biology related disciplines. Through this fellowship, my exposure to knowledge that covers synthetic chemistry, chemical biology of DNA, biophysical characterization, photophysics and microscopy with live cells, has helped me to secure senior postdoctoral position at Kings College London and The Francis Cricks institute in the chemical biology field. Further, the training courses that are offered by PFDC at Imperial contributed significantly, to enhance my knowledge in learning about the innovations, teaching aspects and personal or professional development. The courses, workshop has greatly enhanced my communication skills as well as supervising masters and a PhD student has strengthened my leadership skills, which are fundamental requirements for an independent scientific career. Moreover, Prof. Ramon Vilar has great collaborative network in both industry and academia throughout the world, which helped me expand my international connections in the field of chemical biology. The multidisciplinary research experience that I got through MSCA has strengthen my CV. With this interdisciplinary experience I have applied various top Indian institutes such as IITs (Indian Institute of Technology) and IISERs (Indian Institute of Science Education and Research) and waiting for the interview calls. Overall, I firmly believe that the extraordinary training I got through MSCA can help me to establish myself as independent interdisciplinary researcher in the academia near future.
limitations of previous optical probes such as low brightness, relatively small lifetime range and low selectivity and provide pronounced lifetime differences when it is bound to duplex vs. G4 DNA and led us to image the dynamics of G-quadruplexes in live cells in real time. Although I have developed several cyclometalated Pt-optical probes only one probe has provided PLIM results directly and confirm the ability of the probes to stain G4 quadruplexes within fixed cells. Based on the outcomes of this project it has shown that the potential of cyclometalated Pt-complexes to target G4 DNA structures. Further, this data provides basis for future development of new cyclometalated Pt-complexes with improved cells uptake and ability to visualize G4 DNA in live cells using PLIM.
The other impacts of the projects involve improving the future career prospects of the researcher after the fellowship and effective dissemination of the results obtained in this project. This fellowship has provided me a great opportunity to study and explore the fascinating and emerging research area and make the first steps towards establishing myself as an expert in interdisciplinary research filed. Further, it also helped me in broadening my scientific expertise, strengthen my skill and knowledge in chemical biology related disciplines. Through this fellowship, my exposure to knowledge that covers synthetic chemistry, chemical biology of DNA, biophysical characterization, photophysics and microscopy with live cells, has helped me to secure senior postdoctoral position at Kings College London and The Francis Cricks institute in the chemical biology field. Further, the training courses that are offered by PFDC at Imperial contributed significantly, to enhance my knowledge in learning about the innovations, teaching aspects and personal or professional development. The courses, workshop has greatly enhanced my communication skills as well as supervising masters and a PhD student has strengthened my leadership skills, which are fundamental requirements for an independent scientific career. Moreover, Prof. Ramon Vilar has great collaborative network in both industry and academia throughout the world, which helped me expand my international connections in the field of chemical biology. The multidisciplinary research experience that I got through MSCA has strengthen my CV. With this interdisciplinary experience I have applied various top Indian institutes such as IITs (Indian Institute of Technology) and IISERs (Indian Institute of Science Education and Research) and waiting for the interview calls. Overall, I firmly believe that the extraordinary training I got through MSCA can help me to establish myself as independent interdisciplinary researcher in the academia near future.