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DNA as a training platform for photodynamic processes in soft materials

Periodic Reporting for period 2 - LightDyNAmics (DNA as a training platform for photodynamic processes in soft materials)

Reporting period: 2020-04-01 to 2022-10-31

Light and soft matter interaction is essential to our life and society, being the basis of fundamental biological processes and technological advances. In order to fully master such processes, we have to understand them from the atomic level to the material scale. This is a tremendous challenge and requires the integration of many techniques, from those already existing to those newly in development, incorporating both experimental and computational approaches. The objectives of LightDyNAmics were developing these methods (Work Package 1), using DNA as a training ground (Work Package 2), and applying the knowledge obtained to other multi-chromophoric assemblies, MCA (Work Package 3). The partners of LightDyNAmics (ten leading research institutions, three companies + 12 partner organisations in nine European countries) choose to focus mainly on a single system, to make the transfer of knowledge between the different partners easier, which is why DNA was selected as a training ground. The interaction between light and DNA has a fundamental biological relevance since it can damage genetic code and lead to cellular death and cancer. At the same time, the study of this flexible MCA allows most of the challenges ahead in this field to be tackled. In particular, our research activities aimed to:
1. Develop new time-resolved experimental spectroscopic techniques with both higher temporal resolution and increased sensitivity.
2. Develop new computational methods to allow the simulation of time-resolved spectra, and to describe the excited state dynamics in MCA from femtoseconds to milliseconds.
3. Apply these techniques to representative DNA systems, from nucleobases to quadruple helices, as well as sequences containing newly discovered epigenetic bases.
4. Develop new DNA ligands for diagnostic and therapeutic use.
At the heart of these developments were our 15 early stage researchers (ESRs) who were trained in these multi-disciplinary approaches, cross-developing experimental and computational skills, to become the next generation of scientists.
The beneficiaries recruited the 15 ESRs using a transparent, merit-based procedure, favouring international mobility. Notwithstanding the difficulties due to the outbreak of COVID-19, we attained the main scientific objectives of our network, leading to the publication of more than 50 peer-reviewed articles in world-leading journals, with crucial contributions from our LightDyNAmics ESRs, which also presented their work at a number of international conferences, increasing the outreach of our project (Work Package 5). Our ESRs received key training at the network level (Work Package 4), with five network-wide meetings. Moreover, the ESRs were all enrolled in PhD programmes and received local training from their host institution. They have all successfully completed their Ph.D. studies, with eight already awarded the Ph.D. title. Online training activities have also been implemented, helping to overcome the limitations to the mobility due to COVID-19. Five series of webinars were organised, with fundamental contributions from our ESRs, providing another opportunity to increase their management and dissemination skills. A further key component of acquiring multidisciplinary skills in the network is for the ESRs to perform secondments in other research groups, and 31 of these secondments have been carried out.
Overcoming the difficulties caused by two years of a pandemic, LightDyNAmics thus succeeded in attaining its training and research objectives. 15 ESR have acquired strongly multidisciplinary skills in the study of the interaction between radiation and soft matter, with very good career perspectives. Several of the ESRs have already been granted post-doc positions, both in academia and industry. The main scientific advances are detailed below.
1. We have improved our ultrafast spectroscopy setups to unprecedented time resolution, and made important steps towards 2D UV spectroscopy, an as of yet undeveloped technique. These developments will facilitate the elucidation of the complex spectral signals of MCA.
2. We made important advances in the development of computational methods, improving both our ability in very accurately describing what happens in medium size molecules on an ultrafast time-scale and what happens on a longer time-scale for systems containing many chromophores.
3. The progress in points 1 and 2 allowed a much better understanding of the photophysical processes occurring in the DNA building blocks, the nucleobases, the Watson-Crick hydrogen bonded dimer, and polynucleotides with different arrangements. In particular, we characterized the photophysics of pyrimidines and of several epigenetic derivatives of cytosine, an important achievement in the field of epigenetics.
4. We characterized the primary species (electrons and radicals) resulting by absorption of low energy UV photons by guanine-quadruplexes (GQs), i.e guanine rich regions of DNA that form four stranded quadruple helices, involved in key biological processes. Based on the numerous results collected during the project, we proposed a mechanism explaining this phenomenon. We have also characterized the photoactivated processes in GQs in the presence of binders, either organic or organometallic species. We obtained new insights on the structural dynamics of another DNA arrangement, the Holliday Junction, a key intermediate in many genetic processes. Our studies brought an important insight into the mechanism of oxidative damage to the genetic code, responsible for carcinogenesis and ageing. We hope that they will also benefit the therapeutic efforts in these fields.
5. We have synthesised and studied the mechanism of potential drug scaffolds and a variety of fluorescent probes, likely to be of significant diagnostic and therapeutic use.
6. We started obtaining new information on the interaction between DNA and proteins, i.e. Taq polymerase and two CRISPR associated proteins. These proteins are relevant for two key fields for the research and technological activities of EU. Taq polymerase is indeed frequently used in the polymerase chain reaction (PCR), while CRISPR-Cas system is exploited for genome editing both in the preclinical and clinical settings.
7. The strong expertise the ESRs and the beneficiaries have developed in working on oligonucleotides, as well as in click-chemistry, can have a tremendous impact on the health and medical needs of UE. Two beneficiaries have actively worked in response to the COVID-19 crisis. LMUM has started to use the synthetic oligonucleotide technologies developed in the ETN to design synthetic RNAs that can block virus proliferation, with promising therapeutic potentialities. BAS worked on the production of a lipid nanoparticle-free SARS-CoV-2 vaccine, planned to entry in clinical trials in 2023.
8. Besides their relevance for DNA, the methodological developments attained, together with the insights gained on some ubiquitarian reactions excitation energy transfer (charge transfer, electron ejection), will positively impact the study of the many key biological and technological processes which are driven by the interaction with light.