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Tania’s secondments from Warsaw to Oxford

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Our ESR Tania Sánchez Quirante from the Hocek group at the Institute of Organic Chemistry and Biochemistry in Czechia recently did two secondments at the Universities of Warsaw (Poland) and Oxford (UK).

From January to February 2023, Tania did her first secondment at the Laboratory of Chemical Biology and Biophysical Chemistry at the University of Warsaw, where she synthesized and purified modified cap RNA 70nt long and modified mRNA by in-vitro transcription. She also learned how to do in-vitro translation using the rabbit reticulocyte system.

In April 2023, she stayed at the Tom Brown group at the University of Oxford. She synthesized modified single-guide RNA 99nt long by in-vitro transcription reaction and used this sgRNA to perform CRISPR-Cas experiments. In addition, she did stability experiments of the modified sgRNA in Human serum.

Crystal clear insight: ESRs explore the world of crystallography and structural biology

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The Early-Stage Researchers (ESRs) were thrilled to participate in the 5th training week about nucleic acids and crystallography. The first two days were spent attending the Oligo 2023 conference in Oxford. They listened to experts talk about nucleic acid, base modification, aptamers, and more. They were fascinated by the new developments and ideas in this field.

After the conference, the ESRs visited the Oxford Nanopore Technologies research facilities. They gained a deep understanding of how the research industry works and how scientists turn their ideas into reality. The ESRs were impressed by the state-of-the-art facilities and the advanced equipment used for research.

The next two days were spent at the University of Reading, where the ESRs attended engaging talks about crystallography and data processing. They had the opportunity to participate in hands-on activities such as crystallisation, plate preparation, high throughput robot, and learning to recognise and collect good crystals. Finally, they collected data from the X-ray, which was an exciting experience for them.

After a free afternoon spent in London, the ESRs were welcomed at Diamond Light Source. They were given an intense tour of the different facilities and beam lines available at the synchrotron. They gained an understanding of the Cryo-EM and laser technology used in the field of structural biology for protein and nucleic acids. It was an eye-opening experience for them, and they had a lot of fun learning about this exciting field. Overall, the ESRs found the 5th training week about nucleic acids and crystallography to be an enlightening experience. They were exposed to the latest developments in the field, and the hands-on experience provided them with a deeper understanding of the processes involved. They were inspired to continue their research with renewed enthusiasm and curiosity.

By Ahmad Abdullrahman (ESR4)

Webinar on oligonucleotide synthesis

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Professor Tom Brown (University of Oxford) will be a speaker at the webinar “Traditional and Emerging Methods of Oligonucleotide Synthesis”, organised by Biotage and taking place on the 5th of April at 11am CEST.

Prof. Brown will review the history of oligonucleotide synthesis, examine how they are made currently and highlight emerging synthesis methods, including enzymatic approaches. He will also discuss the various applications of synthetic oligonucleotides, from diagnostics to therapeutics and more.

You can register here: https://go.technologynetworks.com/oligonucleotide-synthesis

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

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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

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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.

Eva’s secondment at the Kellett Group in DCU

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baseclick GmbH and the Kellett group at DCU have an ongoing research collaboration upon a potential oligonucleotide-based SARS-CoV2 drug, whereas our baseclick ESR Eva Sophie Schönegger, one PostDoc (Dr. Brionna McGroman) and one PhD Student from the Kellett group are working together.

During the secondment in October 2022, Eva learned new techniques and developed new skills. At DCU, Eva was analyzing binding affinities of ligand-clicked oligonucleotides, which she had previously prepared in Munich. Therefore, different approaches were investigated, based on thermal melting, microscale thermophoresis and gel electrophoresis. Moreover, she performed first cleavage assays with the best-performing oligonucleotides

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

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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

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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

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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.