Proof of Concept for a Platform for the Fast and General Elucidation of the Interaction Between Proteins and RNA

Project Information

ProteRNA

Grant agreement ID: 101006134

DOI

10.3030/101006134

Project closed

EC signature date 24 November 2020

Start date 1 July 2021

End date 31 December 2024

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 257 600,00

EU contribution

€ 257 600,00

Coordinated by

KELADA PHARMACHEM LIMITED
Ireland

Periodic Reporting for period 2 - ProteRNA (Proof of Concept for a Platform for the Fast and General Elucidation of the Interaction Between Proteins and RNA)

Reporting period: 2024-01-01 to 2024-12-31

Summary of the context and overall objectives of the project

Proteins which have been found to have a genetically verified link to diseases are twice as likely to be therapeutically validated as drug targets when compared to those without any genetic link to disesase.1 In this regard, the amazing progress which has been made in mapping the human genome and proteome has led to significant hope for the development of novel therapies which regulate expression of proteins related to diseases. However, most studies in this area lack an understanding of the functional and regulatory relationships between proteins and gene expression to deliver such therapies. The impediment of developing such knowledge is the lack of effective analytical tools which can map the interactions of cellular RNA-binding proteins (RBPs) and oligonucleotides (e.g. RNA).
The development of analytical methods for identification of RNA-binding proteins (RPBs) and their corresponding RNA motifs, will facilitate: i) a deeper understanding of the roles of gene regulation and protein expression; ii) accelerate development of personalised medicines via regulation of protein expression; and iii) enable rapid identification of proteomes involved in specific diseases. In doing so, this project will have outputs which in the medium term will have significant market opportunities and societal benefits, including, but not limited to, stimulating job creation in the pharmabiotech and diagnostics sectors; and in the longer term reducing costs for national and EU healthcare budgets.

Against the above background, this project seeks to address the intertwined challenges of helping to grow the European biotechnology and pharmaceutical industries via new technology and product innovation, whilst at the same time ensuring that such technologies and products can ultimately be developed (and implemented) economically.
We will do this by tackling the following specific R&I objectives:
Objective 1 – Develop a novel method for immobilising oligonucleotides (RNA) onto solid support using 19F-tagged linkers
Objective 2 – Test and confirm that complexation of the immobilised RNAs with known RBPs can be validated by 19F-NMR spectroscopy and mass spectrometry
Objective 3 – Test and confirm that the RNA-RBP complex analysis platform can identify unknown RBPs from proteic fractions of cell lysates

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The work in this project is to develop and establish proof-of-concept of the utility of a new platform for fast and general elucidation of the interaction between proteins and RNA.

This work involves:
• KelAda developing a protocol for the immobilisation of RNAs using a linking scaffold with four key functional aspects: i) a terminal N-hydroxysuccinimide ester motif for linking to amine-functionalised surfaces via amide bond formation; ii) a terminal alkyne motif for linking to azide-functionalised RNA strands via click reaction; iii) a fluorinated motif which will act as an indicator for RNA-RBP complexation via 19F-NMR spectroscopy; and iv) a photocleavable nitrobenzyl motif which will permit light-stimulated liberation of immobilised 19F-tagged RNA-RBP complexes.
• CEITEC developing benchmark validation of interaction of immobilized RNAs with RBPs by 19F RNA. RNAs of known structure and a complementary protein which binds to that RNA, will form the basis for determining the benchmark NMR assay.
• AUTh developing a protocol for the identification of novel RNA-binding proteins by mass spectrometry and to quickly characterise the mode of interaction of these novel RNA-RBP complexes by NMR spectroscopy.

Due to the Covid 19 pandemic and associated laboratory closures and travel restrictions there was a delay in initiating some aspects of the project. Appropriate risks were identified and mitigation measures were put in place. There were also issues recruiting suitable secondees. As a result an amendment to the grant was sought and approved. An additional 6 months was added to the time period of the project.
The main impact was on WP1 deliverable D1.1 which had a resultant impact on WP2 and WP3. Strategies were devised to overcome this issue.
Training of secondees in their Host Institution continued. Alternative methods for immobilising RNA were examined as proof-of-concept experiments. Work included preparation and analysis of complex biological samples beginning with a commercially available product while awaiting the WP1 deliverables. An alternative route for the synthesis of the product of D1.1 was devised and is ongoing.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Biophysical and biochemical analytical methods developed over the past 60 years have driven discoveries in biology and in the last 20 years especially in RNA biology. Only 2% of the genome codes specifically for proteins, meanwhile a vast amount of RNAs (ca. 290.000) are produced that exert a function in the cell by interacting with proteins. With only 25% of proteins known to be druggable, RNA is the logical next drug target. ProteRNA constitutes a platform for studying RNA-protein interactions and is therefore timely but also has a great potential to contribute substantially in the endeavour to understand the RNA-protein interactome and its role in disease. The most commonly used methods fulfil the scope of isolating RBPs, however they involve extensive laboratory and manual operation to complete the task. The current state of art permits the isolation of only a limited (mRNA) subset of protein-RNA complexes, which are formed via chemical RNA modification and rely on laborious procedures, including RNA internalisation and its photochemical reaction with RBPs followed by isolation of covalently bound RNA-RBP constructs. The consortium have identified an opportunity to address the gaps in the state of the art and thus improve the understanding of the functional and regulatory aspects of the RNA-binding protein (RBP) interactome, which would lead to the development of novel therapies and diagnostics. We have therefore identified an opportunity to develop and prove the concept for an analytical platform which can allow i) the rapid validation of specific RNA-RBP interactions and ii) elucidate the nature of those interactions, leading to mapping of specific RNA-RBP interactomes in a fast and general way.

EU economic growth and the sustainability of world-class companies and academic institutions will be directly impacted by the quality of research being performed in this project. In turn, this depends upon excellent and long lasting intra- & inter-sectoral and international collaboration. In bringing KelAda from the commercial sector, together with AUTh and CEITEC-MU from academia, ProteRNA will play a significant role in bettering the integration of European science and thus yielding a long-term economic impact. By leveraging the inherent and differentiating benefits of the ProteRNA RNA-ligand analysis platform, we will significantly add to Europe’s competitiveness in drug development/pharmaceutical research and to maintain a leading global position in the areas of drug and diagnostic research & development.

fig-1-state-of-the-art-discoveries-and-therapies-for-rna-and-rnabp.png

fig-2-proterna-linker-immobilisation-scheme.png