DCU and LMU publish review on Third Generation Sequencing of Epigenetic DNA

The Kellett (DCU) and Carell (LMU) groups recently published a collaborative review titled ‘Third Generation Sequencing of Epigenetic DNA’ in Angewandte Chemie. The review is open access and covers the latest developments in sequencing techniques adapted and developed for ‘third generation’ sequencing platforms, which promise to provide the fastest and most convenient means of DNA sequencing to date.

Cytosine modifications have been shown to influence gene regulation, in turn effecting disease and development, thus facile methods for sequencing these base modifications by exploiting the chemistries of these new devices is an active area of research. Despite extensive reviews covering sequencing technologies and base modifications independently, to our knowledge, this is the first publication to highlight the emerging potential of third generation sequencing technologies to expedite epigenetic research.

During the first in-person NATURE-ETN training week organised in the Institute for Chemical Epigenetic – Munich (ICE-M), Dr Markus Müller and Dr. Pascal Giehr delivered seminars focused on epigenome sequencing which provided valuable background in techniques developed to facilitate the decoding of this secondary information layer in DNA. Work Package 3 in NATURE-ETN aims to generate new techniques for sequencing and imaging epigenetic bases. DCU and LMU have access to third generation sequencing devices, so this review will provide a helpful reference point for researchers in the network.  

Eva publishes about click chemistry-based library preparation for long-read third-generation sequencing

The communication paper of ESR Eva Schönegger and Dr. Antony Crisp from baseclick GmbH in collaboration with the LMU Munich, Institute for Chemical Epigenetics Munich was published in September 2022 in Bioconjugate Chemistry.

In this communication paper, entitled Click Chemistry Enables Rapid Amplification of Full-Length Reverse Transcripts for Long-Read Third Generation Sequencing, Eva, Dr. Antony Crisp, Dr. Markus Müller and coworkers describe the development of a novel click chemistry-based method for the generation and amplification of full-length cDNA libraries from total RNA.

In this work, supervised by Prof. Thomas Carell (LMU) and Dr. Thomas Frischmuth (baseclick), the use of click chemistry circumvents the need for the problematic template-switching reaction.

The use of PCR primers containing two overhanging 3′-nucleotides is one essential modification of the described workflow resulting in a significantly improved read-through compatibility of the 1,4-disubstituted 1,2,3-triazole-containing cDNA, where these modifications normally hinder amplification. This enables to use an insert size which is twice as large compared to the state-of-the art click chemistry-based technique, PAC-seq.

Taking the known advantages of PAC-seq, such as suppression of PCR artefacts, into consideration, the described library preparation method could enable various applications, including improved analyses of mRNA splicing variants and fusion transcripts.

The 4th training week – In the core of NATURE-ETN

Early-stage researchers (ESRs) and principal investigators have been invited to attend the 4th training week at Dublin City University where this EU-funded research consortium was born.

Prof. Andrew Kellett first gave an update on the project before introducing Prof. Nicholas Farrell from Virginia Commonwealth University (US), who is one of the scientific advisors of NATURE-ETN and is interested in Glycosaminoglycans as signalling molecules. He talked about his latest results and joked about how retiring is difficult when you can’t stop looking for more results.

Each ESR presented their results obtained so far followed by questions, comments, and discussions for future ideas. On Thursday, a team from the German biophysical instrument manufacturer NanoTemper delivered training on the Monolith technologies in Andrew Kellett’s lab to ESRs who had the opportunity to prepare the samples, learn how to read the results, and think about how this technology can be applied to their projects.

Finally, on the last day of the training week, ESRs were hosted by Prof. Niall Barron at the National Institute for Bioprocessing Research and Training (NIBRT). Prof. Barron explained the advanced therapy medicinal products, emphasizing the need to keep in mind the patient delivery process while obtaining basic research results. ESRs conducted a complete tour guided by Dr Adam Pritchard who showed and explained the technologies behind the bioproduction of recombinant proteins, vaccines, and cell and gene therapies.

Before saying goodbye, the ESRs decided to go for a walk together. This time down to the lighthouse at Dún Laoghaire, a small town outside the busy Dublin. Prof. Barron mentioned Emerson who said, “Science does not know its debt to imagination”, and isn’t the sea the best place to clear the mind and fire the imagination?

In this training week, new collaborations were created between ESRs, wrapping up before departing to their different locations in Europe, where they will bring home new ideas to apply in their labs and Irish souvenirs!

By Ahmad Abdullrahman (ESR4)

Jamie’s secondment at the MRC Laboratory of Molecular Biology completed

Our ESR Chun Yin (Jamie) Chan from the Carell group at LMU had the opportunity to go on secondment at the MRC Laboratory of Molecular Biology (LMB) in Cambridge, UK, from July to October 2022.

The MRC LMB is one of the world’s leading research institutes in molecular biology. Jamie joined the group led by Dr. Philipp Holliger, which focuses on chemical biology, synthetic biology and in vitro evolution. The Holliger lab investigates fundamental questions of the chemical logic and constraints of molecular information encoding and the encoded synthesis; replication and evolution of novel sequence-defined biopolymers for applications in biotechnology and medicine; and the emergence of genetic function in chemical systems.

During his stay, Jamie explored the capacity of a recently discovered RNA polymerase ribozyme utilising trinucleotide triphosphates (triplets) as substrates to incorporate chemically modified triplets, for example, for the template-directed and sequence-specific positioning of chemical groups in nucleic acid polymers, and investigated how could these modifications possibly impact the ribozyme’s polymerisation in terms of fidelity, activity and chemical space for evolution in a prebiotic scenario.

NATURE-ETN ESRs publish review on DNA triplexes

A collaborative review by ESRs Ahmad Abdullrahman from the Department of Pharmacy, Chemistry and Pharmacy Building, University of Reading (UK), and Maria Dalla Pozza from Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences (France) was published in Chemical Science in August 2022.

In this review, entitled Three’s a crowd – stabilisation, structure, and applications of DNA triplexes, the ESRs put their efforts together in proficient teamwork supervised by Dr. James Hall, Prof. Christine Cardin from the University of Reading, and Prof. Gilles Gasser from Chimie ParisTech, PSL University.

They describe the main characteristics of triplex DNA structures, focusing on their application and interaction with metal compounds, highlighting the need for additional structure characterization and biological studies.

The DNA triplex may be formed naturally, during homologous recombination, or can be formed by the introduction of a synthetic triplex-forming oligonucleotide (TFO) to a DNA duplex. Among others, the most interesting feature of TFOs is their sequence specificity in binding a duplex DNA. The authors first compare the triplex structure with the canonical B-DNA structure. Subsequently, they report the main modifications at the base, sugar and phosphate backbone levels currently available to obtain a more stable structure, considering the potential in vivo conditions.

There is significant interest in developing TFOs with potential therapeutic applications, including their use as a delivery mechanism for compounds able to modify or damage DNA. However, to combine triplexes with functionalised compounds, a full understanding of triplex structure and chemical modification strategies is essential, stress the authors. In the review, these areas are discussed. Moreover, the possible use of photoactive Ruthenium polypyridyl complexes as a suitable photophysical payload for a TFO system is presented in this scientific paper. With the hope that future research will harness the peculiar characteristics of DNA triplexes.

Third NATURE-ETN training Week in Munich, Germany

From the 16th to the 20th of May 2022, our fifteen NATURE-ETN early-stage researchers (ESRs) were finally reunited in Munich, Germany, for the third training week. The event was held in the recently built Institute of Chemical Epigenetics Munich (ICEM), in the Ludwig-Maximilian University of Munich (LMU). The ICEM building is funded by the Federal Republic of Germany and the state of Bavaria and results from the initiative of Prof. Thomas Carell, Principal Investigator in NATURE-ETN. It hosts two of our ESRs from the Carell group who recently moved to this new building.

On the first day of the training week, the ESRs received a guided tour of ICEM and introductory sessions on mass-spectrometry-based proteomics by Dr. Pavel Kielkowski and sequencing technology by Dr. Markus Müller.

On the second day, the scientific training was pursued with morning sessions on epigenome sequencing by Dr. Müller and Dr. Pascal Giehr. In the afternoon, Dr. Müller provided a bioinformatics crash-course with RNAseq and Prof. Carell gave a lecture on the synthetic chemistry of nucleosides.

On the third day, the ESRs formed groups to discuss ways of enhancing science communication to different audiences.

The training programme also covered industry-relevant skills, with a stimulating session on Quality Management delivered by Alexander Schalk, Quality Manager at the company Baseclick GmbH, also a beneficiary in NATURE-ETN.

The ESRs had the afternoon free to visit Munich and better get to know each other, in the first in-person meeting since the start of the project.

On the fourth and fifth days, the ESRs, along with other researchers from the LMU Department of Chemistry, attended the Nature masterclass delivered by Dr. Véronique Gebala, Senior Editor at Nature, and Dr. Ilse A. Valtierra-Gutierrez, Associate Editor at Nature Communications.

The comprehensive training included sessions on the preparation and submission of high-quality papers, the editorial and peer-review processes, and best practices in scientific publishing. The trainers also take the time to review abstracts prepared in advance by the ESRs.

We thank the organisers at LMU and the trainers for this successful event and already look forward to the 4th training week, to be held this Autumn in Dublin, Ireland.

In the meantime, most ESRs will meet again on the 5th to 10th of June for the XVIIIth Symposium on Chemistry of Nucleic Acids, in Český Krumlov, Czechia, organised by the Hocek group.

From Paris to Reading – Maria’s secondment in the UK

During my NATURE-ETN PhD program, I had the chance to spend two months at the University of Reading, in the UK, supervised by Prof. Christine Cardin and Dr. James Hall.

Even if changing country and environment are always challenging, I realised how a great experience I had the chance to come across from the first moment I arrived in the UK. The first day I went to the lab, the group gave me a very warm welcome, and I felt at home from the beginning.

During this secondment, I learnt many new techniques and developed new skills, supervised by very professional and passionate scientists, always keen to teach me.

During these two months, I evaluated the interaction between the compounds I synthesised at Chimie ParisTech and the DNA, through thermal stability studies. Also, I learned how to use a chiral HPLC and crystallise the compounds and the DNA. These topics and techniques are really fascinating to me, and this experience helped me to better understand my project and its very important aim. Overall, during my secondment, I had the opportunity to learn, develop my scientific skills, and know a different scientific environment.

To be fair, that was not only work… I fell in love with the UK! During the weekend I visited many different places like London, Oxford, and the beautiful British countryside. I spent some evenings in the pubs drinking beers with my new friends and having tea with the delicious scones with clotted cream and jam (the “cream tea”) in the afternoon.

Also, we went out with the group for a beer a few times, as you can see in the picture.

This was a very great experience from both a professional and personal point of view, and I am grateful to be part of this fantastic ETN project.

By Maria Dalla Pozza (PSL)

New publication on the cytotoxic activity of organometallic di-iron complexes in cancer cell lines

Recent collaborative work from NATURE-ETN and the University of Pisa has been published in Organometallics by ESR Maria Dalla Pozza from the group of Gilles Gasser at Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences (France), in collaboration with the team of Prof. Fabio Marchetti at the University of Pisa (Italy). The scientists investigated the anticancer activity of some di-iron compounds functionalised with sugar moieties to increase their cellular uptake and possibly their selectivity for cancer cells. The compounds, synthesised by Marchetti’s group, were tested against a panel of cancerous cells by ESR Maria Dalla Pozza.

Among the different categories of metal drug candidates, iron complexes based on the ferrocene scaffold have aroused notable interest. The antiproliferative activity of these compounds is ascribable to the redox chemistry of the ferrocenyl iron(II) center, which undergoes oxidation to Fe(III) in the tumour cells. This enhances the formation of toxic metabolites that lead to cell death. Di-iron compounds have been less investigated, however recent studies have disclosed a promising anticancer potential. In this study, the sugar functionalisation was performed on di-iron complexes to optimise the cytotoxic properties by exploiting the Warburg effect. Specifically, the anticancer activity of the compounds was evaluated using the resazurin assay and the scratch assay. Overall, this study pointed out that the antiproliferative activities of the new complexes correlate with their lipophilicity, ranging from moderate cytotoxicity to inactivity, and showing an absence of appreciable selectivity with respect to a non-cancerous cell line. On the other hand, analogous diiron complexes without the sugar moiety functionalisation, analysed as references, performed better in the same conditions. This confirms the potential of the present category of organometallics in the medicinal field.

Reference:
Schoch, S., Iacopini, D., Dalla Pozza, M., Di Pietro, S., Degano, I., Gasser, G., Di Bussolo, V., and Marchetti, F. Tethering Carbohydrates to the Vinyliminium Ligand of Antiproliferative Organometallic Diiron Complexes. 
Organometallics (2022). https://doi.org/10.1021/acs.organomet.1c00519

New publication examines the multi-modal activity of copper(II) and silver (I)-phendione complexes on DNA scission within P. aeruginosa

Recent collaborative work from NATURE-ETN has been published in the Journal of Biological Inorganic Chemistry by researchers in the Kellett lab at DCU. Co-authors include NATURE-ETN coordinator Dr. Andrew Kellett, co-supervisor Dr. Georgia Menounou, and ESR Conor Bain. The paper investigated the multi-modal activity of copper(II) and silver(I) complexes with the N,N-coordinating ligand, 1,10-phenanthroline-5,6-dione, with particular focus on DNA damage within Pseudomonas aeruginosa.

The emergence of microbial drug-resistance in recent decades has given rise to the need for novel antimicrobial therapeutics. The metal-based complexes [Ag(1,10-phenanthroline-5,6- dione)2]ClO4 (Ag-phendione) and [Cu(1,10-phenanthroline-5,6-dione)3](ClO4)2.4H2O (Cu-phendione) have previously demonstrated efficient antimicrobial action against multidrug-resistant species. The focus of the study was to understand the binding potential of these complexes with double-stranded DNA using a combination of in silico and in vitro approaches. Promising results arising from this work revealed a potentially new class of antimicrobial drug candidate with a distinct therapeutic mechanism against the multidrug-resistant pathogen P. aeruginosa.

Molecular docking studies showed both complexes elicit a multi-mechanistic approach to DNA-binding via hydrogen bonding, hydrophobic and electrostatic interactions, with both complexes favouring minor groove binding. Of the two complexes, Cu-phendione achieved the highest binding affinity for both major and minor grooves with nearly 10x greater affinity to DNA than Ag-phendione and nearly 20x greater affinity than the phendione ligand alone. Cu-phendione achieved DNA scission through free radical oxidative damage, as well as DNA-nicking and relaxation of supercoiled plasmid DNA. It was concluded that both complexes elicit a dose-dependent effect, with successful DNA fragmentation within multi-drug resistant pathogen P. aeruginosa when treated with a single dose of Cu-phendione. This work proposes a novel dose-regulated class of metal-based antimicrobial therapeutics.

Reference:

Galdino, A.C.M., Viganor, L., Pereira, M.M., Devereux, M., McCann, M., Branquinha, M.H., Molphy, Z., O’Carroll, S., Bain, C., Menounou, G., Kellett, A., Dos Santos, A.L.S. Copper(II) and silver(I)-1,10-phenanthroline-5,6-dione complexes interact with double-stranded DNA: further evidence of their apparent multi-modal activity towards Pseudomonas aeruginosaJ Biol Inorg Chem (2022). https://doi.org/10.1007/s00775-021-01922-3