Periodic Report Summary 2 - LIGHTSWITCHES (Multifunctional DNA light-switches: sensors and devices)
Briefly, the aims of this project were:
(i) The synthesis of mononuclear complexes with tuneable luminescent and/or photo-redox properties will be targeted.
(ii) To synthesise 'light-switch' systems designed to display selectivity on binding with DNA.
(iii) Strategies designed to enhance overall binding affinity of mixed bis-intercalators will be investigated. Such systems will be synthesized as tools to study DNA mediated energy transfer processes.
(iv) The synthesis of multifunctional DNA probes was also investigated.
Tuning photophysical properties of DNA binding complexes, by using a variety of different intercalating ligands and non-intercalating ancillary ligands, the physical properties of our intercalative systems have been tuned. For example, although we have previously reported that dppz-complex, is a simple 'light-switch' complex that possesses an excited state incapable of cleaving DNA, work during this project has revealed that the photoexcited state of the related dppn-based complex is a highly efficient singlet oxygen sensitizer. Singlet oxygen IS capable of damaging and cleaving DNA. By changing the donor / acceptor capability of the ancillary ligands, we have tuned the emission wavelength from ~600 nm to ~680 nm.
Tuning binding selectivity of the complexes
Studies on our prototypical quadruplex DNA binding agent, has revealed that the complex binds to specific quadruplexes with specific structural features: it binds with highest affinity to quadruplexes with diagonal external loops. The binding intensity is also reflected in the emission response, with the highest luminescence enhancements observed for the structures that are bound with the highest affinity.
Approaches to sequence selectivity have also been developed; for example, by adding hydrogen-bonding units to the pyridyl ancillary ligand, we have been able to create systems that show binding preferences for A- or G-rich DNA sequences. We have even been able to switch off intercalation at together.
Bis-intercalators
We have found we can enhance binding of our dinuclear complexes by a consideration of the linkers employed. For example by using linkers that contain amines, we have been able to enhance the overall binding affinity of the systems by 1-2 orders of magnitude.
Multifunctional probes
Perhaps our key results have been to begin to study the interaction of such systems in cellulo. In these studies we have found that while 3 is a live/dead stain, is a multifunctional probe for cellular imaging. Using confocal microscopy, CFM, it can be used to image duplex DNA-based heterochromatin, for example individual chromosomes can be imaged during mitosis. However, at a different emission wavelength the same complex is a marker for higher order DNA structures within cells. Finally, because the complex incorporates heavy ruthenium nuclei, it is also a novel contrast stain for transmission electron microscope, TEM, which can be used to image nuclear DNA. Furthermore, in recent work we have found that the related mononuclear complex, is not only an imaging agent for cells through CFM and TEM, but it is also displays a cytotoxicity that is comparable to cisplatin, and this cyctotoxicity is retained even in cisplatin-resistant cell-lines.
Several publications in high impact journals and a patent application have resulted from this project. A second patent application is currently being drafted. The potential impact of this work is high: therapeutic leads for cancer treatment have been identified and a new paradigm for cell imaging has been developed.
(i) The synthesis of mononuclear complexes with tuneable luminescent and/or photo-redox properties will be targeted.
(ii) To synthesise 'light-switch' systems designed to display selectivity on binding with DNA.
(iii) Strategies designed to enhance overall binding affinity of mixed bis-intercalators will be investigated. Such systems will be synthesized as tools to study DNA mediated energy transfer processes.
(iv) The synthesis of multifunctional DNA probes was also investigated.
Tuning photophysical properties of DNA binding complexes, by using a variety of different intercalating ligands and non-intercalating ancillary ligands, the physical properties of our intercalative systems have been tuned. For example, although we have previously reported that dppz-complex, is a simple 'light-switch' complex that possesses an excited state incapable of cleaving DNA, work during this project has revealed that the photoexcited state of the related dppn-based complex is a highly efficient singlet oxygen sensitizer. Singlet oxygen IS capable of damaging and cleaving DNA. By changing the donor / acceptor capability of the ancillary ligands, we have tuned the emission wavelength from ~600 nm to ~680 nm.
Tuning binding selectivity of the complexes
Studies on our prototypical quadruplex DNA binding agent, has revealed that the complex binds to specific quadruplexes with specific structural features: it binds with highest affinity to quadruplexes with diagonal external loops. The binding intensity is also reflected in the emission response, with the highest luminescence enhancements observed for the structures that are bound with the highest affinity.
Approaches to sequence selectivity have also been developed; for example, by adding hydrogen-bonding units to the pyridyl ancillary ligand, we have been able to create systems that show binding preferences for A- or G-rich DNA sequences. We have even been able to switch off intercalation at together.
Bis-intercalators
We have found we can enhance binding of our dinuclear complexes by a consideration of the linkers employed. For example by using linkers that contain amines, we have been able to enhance the overall binding affinity of the systems by 1-2 orders of magnitude.
Multifunctional probes
Perhaps our key results have been to begin to study the interaction of such systems in cellulo. In these studies we have found that while 3 is a live/dead stain, is a multifunctional probe for cellular imaging. Using confocal microscopy, CFM, it can be used to image duplex DNA-based heterochromatin, for example individual chromosomes can be imaged during mitosis. However, at a different emission wavelength the same complex is a marker for higher order DNA structures within cells. Finally, because the complex incorporates heavy ruthenium nuclei, it is also a novel contrast stain for transmission electron microscope, TEM, which can be used to image nuclear DNA. Furthermore, in recent work we have found that the related mononuclear complex, is not only an imaging agent for cells through CFM and TEM, but it is also displays a cytotoxicity that is comparable to cisplatin, and this cyctotoxicity is retained even in cisplatin-resistant cell-lines.
Several publications in high impact journals and a patent application have resulted from this project. A second patent application is currently being drafted. The potential impact of this work is high: therapeutic leads for cancer treatment have been identified and a new paradigm for cell imaging has been developed.