Illuminating aptamers and ribozymes for biomolecular tagging and fluorogen activation

Project Information

illumizymes

Grant agreement ID: 682586

DOI

10.3030/682586

Project closed

EC signature date 5 April 2016

Start date 1 July 2016

End date 30 June 2022

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 061 250,00

EU contribution

€ 2 061 250,00

Coordinated by

JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG
Germany

Periodic Reporting for period 4 - illumizymes (Illuminating aptamers and ribozymes for biomolecular tagging and fluorogen activation)

Reporting period: 2020-08-01 to 2022-06-30

Summary of the context and overall objectives of the project

The main goal of the ERC Consolidator Grant „Illuminating Aptamers and Ribozymes for Biomolecular Tagging and Fluorogen Activation“ (ILLUMIZYMES) aimed at the development of new classes of synthetic nucleic acid analogs as versatile and efficient tools for molecular imaging and the functional characterization of natural RNAs and their modifications. Despite the relative simplicity in molecular composition, the available methodological repertoire for manipulation, interrogation, and visualization of RNA still is rather limited. The major research directions in the project were devoted to the exploration and exploitation of the catalytic repertoire of nucleic acids in the laboratory with the fundamental goal to identify new nucleic acid enzymes (ribozymes and deoxyribozymes) by in vitro selection from random RNA and DNA libraries, a process that can be fine-tuned to identify catalytic activities of DNA and RNA molecules that have so far not been found in nature. In addition, we focussed on the development of RNA aptamers (FLAPs) as fluorescent reporters for tagging and visualization of RNA in complex environments. By capitalizing on the progammable nature of tools based on nucleic acids, and by encoding the activity in the sequence and structure of nucleic acid catalysts, we managed to develop a series of novel-type chemical probes termed illumizymes. A major focus of the project was on the in vitro selection and characterization of new (deoxy)ribozymes for site-specific labeling of RNA and on the evolution of functional nucleic acids for the detection of nucleoside modifications. The tools developed in this project also include fluorogenic aptamers and reversilbly color-switchable illumizymes. The resulting catalysts are promising and versatile tools for nucleic acids research and beyond. The new genetically encodable RNA devices can be engineered into tags and sensors for proteins and small molecules, and will find widespread applications to enlighten our understanding of cellular RNA functions in health and disease.

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

A major focus of the project was on the in vitro selection and characterization of new ribozymes for site-specific labeling of RNA and on the evolution of functional nucleic acids for the detection of nucleoside modifications. The development of the first known methyltransferase ribozyme catalyzing the site-specific installation of 1-methyladenosine in a substrate RNA, using O6-methylguanine as a small-molecule cofactor, was a real milestone within the project and can be regarded as a true landmark discovery (published in Nature, see also next paragraph). In addition, we succeeded in solving the structure and the mode of action of MTR1. The crystal structure of the methyltransferase ribozyme reveals a guanine-binding site reminiscent of natural guanine riboswitches. Together with the surprising similarity between our laboratory-evolved ribozyme MTR1 and naturally occurring RNA motifs, our results provide important support for the so-called RNA world hypothesis giving further evidence that RNA could have been one of the first information-storing and catalytically active polymers.
Also, we characterized a large Stokes shift fluorogenic RNA aptamer named Chili that binds the ligands in the protonated phenol form and exploits excited state proton transfer pathways to enable a more than 350-fold enhanced fluorescence emission from the phenolate form of the bound chromophore. The ligands feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. Our results suggest that Chili might be a versatile tool for future imaging applications.
Altogether, we managed to achieve many impressive results that already led to 19 publications in renowned, high-ranking journals such as Nature, Nature Chemical Biology, Nature Communications, Nature Structural & Molecular Biology, Angewandte Chemie (among them a VIP Paper and three Hot Papers), and The Journal of the American Chemical Society. Further manuscripts are currently in preparation. Some of our findings can be regarded as important landmarks for future investigations (see next paragraph). Furthermore, we presented many of our exciting discoveries to the scientific community at important conferences, in invited key lectures given by the PI, and in oral and poster presentations given by the co-workers involved in the project. The summer school on nucleic acid chemistry and synthetic biology in 2019, offering lectures from renowned experts on a wide variety of topics around the chemistry of nucleic acids, was an ideal platform for networking and the exchange of research ideas and experience related to our project. Overall, the funding that was granted made it possible to achieve many trend-setting results and led to increased visibility of our research group.

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)

Significant achievements have been made within the project, with results far exceeding initial expectations. The discovery of the very first methyltransferase ribozyme, named MTR1, by in vitro selection from random nucleic acid libraries and the elucidation of its exact 3D structure including its mode of action was a scientific breakthrough. This finding can be taken as further evidence for the "RNA world hypothesis", postulating that methylated RNAs played a crucial role on Early Earth in maintaining and improving vital cellular functions. New perspectives will arise from this finding and the newly developed ribozyme is expected to be a useful tool for a wide variety of research areas in the future. In particular, MTR1 will contribute to a better understanding of the interaction of methylation, structure, and function of RNAs and will encourage the in vitro evolution of further ribozymes catalyzing a variety of other reactions.
A new high-throughput approach was developed in the project, named DZ-seq. The innovative character of the new approach becomes apparent from the fact that DZ-seq is applicable for comprehensive studies on huge combinatorial libraries and can also be used for the analysis of very different types of nucleic acids. Since the new profiling approach makes it possible to identify deoxyribozymes that would have remained inaccessible using conventional methods, it will largely expand the portfolio of catalytic nucleic acids as powerful tools for biochemical research.
The fluorogenic Chili RNA aptamer attracted attention as an versatile and efficient RNA mimics of fluorescent proteins that specifically bind and activate conditional fluorophores such as analogs of the green and red fluorescent protein chromophore 4-hydroxybenzylidene imidazolone (HBI). Chili exclusively binds the protonated phenol form of the HBI derivatives and exploits excited state proton transfer pathways to enable a more than 350-fold enhanced fluorescence emission from the phenolate form of the HBI chromophore. The elucidation of the structural and mechanistic basis of fluorescence activation in the Chili aptamer ligand complex will pave the way for future engineering of fluorogenic moduls for sensors and imaging applications.

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