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Development of photoswitchable ligands for the reversible and selective assembly with RNA

Periodic Reporting for period 1 - PHOTORNA (Development of photoswitchable ligands for the reversible and selective assembly with RNA)

Reporting period: 2017-10-01 to 2019-09-30

The ribonucleic acid (RNA) represents an important target for therapy of viral and bacterial infections, a range of genetic diseases and cancer. However, due to the structural diversity of RNA, the rational design of selective RNA binders remains a highly challenging task. Another problem of the development of nucleic acid-targeting drugs is the realization of spatial and temporal control of their activity. From this point of view, manipulation of the drug activity by light is a highly attractive approach. However, the field of photocontrolled RNA-binders has been unexpectedly neglected. Up to now, photoinduced association of small molecules with RNA has been demonstrated only for a few examples. Therefore, the main objective of this project was the development of novel photoswitchable RNA binders whose properties can be reversibly switched and controlled by light. Additionally, the present project was focused on the interactions of photoswitchable ligands with naturally occurring RNAs possessing distinct biological function, so that these studies aimed to reveal novel, medically relevant insights.
Within the WP1, design and synthesis of photoactive hemi-(thio)indigo and fulgide/fulgimide derivatives was performed. Due to significant synthetic difficulties the expected fulgide/fulgimide derivatives could not be obtained. The hemi(thio)indigo derivatives were synthesized but they showed too low solubility in water and low thermal stability of the photoinduced form. Therefore, further the research was focused on hemi-indigo compounds. Thus, a range of hemi-indigo derivatives was synthesized and their photoswitching in water was tested. The compound with best photoswitching characteristics in aqueous medium was identified and further chemically modified to provide the required affinity towards RNA. Several modification patterns have been checked to find the most appropriate one. Additionally, the hemi-indigo dimers comprising two photoswitchable units were designed and synthesized. The compounds showed interesting photoswitching properties in organic solvents but their low solubility in water precluded further studies with RNA.
Within the WP2, the selection of the most structurally fitting naturally occurring RNA sequences was performed. Two elements of the human immunodeficiency virus (HIV) genome RNA were chosen as targets for the binding with a photoswitchable hemi-indigo ligand, namely, trans-activation response RNA (TAR) and Rev-response RNA stem IIB (RRE-IIB). These RNA motifs have a therapeutic importance for the treatment of HIV infection. Binding of the hemi-indigo ligand to TAR and RRE-IIB RNA and its photoswitching in the ligand–RNA complexes were studied by a variety of spectroscopic and bioanalytical methods. It was found that the fluorescence of the ligand in the presence of RNA can be reversibly switched ON and OFF by light and the fluorescence light-up effect was strongly dependent on the RNA sequence. These properties make hemi-indigo derivatives promising candidates for the application in real-time monitoring of biological systems. Additionally, the extended photochemical studies of hemi-indigo dimers in organic solvent were performed using the real-time NMR spectroscopy with in-situ irradiation.
Overall, the hemi-indigo scaffold was introduced as a platform for a novel class of photoswitchable RNA binders possessing valuable features for binding and controlled detection of viral RNA and RNA–protein complexes that can be used in diagnostics and treatment of a range of diseases.
The results of the project were published in two papers in the international peer-reviewed journals, presented at three international conferences as well as disseminated to the broader audience through the outreach events.
Within this project, the following main contributions to the state of the art were made:
• First example of the interaction of hemi-indigo photochromes with nucleic acids – introduction of a novel class of RNA binders.
• First study of the photoswitching in the presence of the natural therapeutically relevant RNA (HIV RNA).
• Proof-of-principle of the application of hemi-indigo derivatives as RNA stains with photoswitchable fluorescence.
• Development of the real-time nuclear magnetic resonance (NMR) spectroscopy for monitoring of the photochemical reactions of hemi-indigo derivatives.

The results of this project contributed to the strengthening of science in Europe in the area of nucleic acids chemistry, particularly in the development of lead structures for RNA-targeting drugs and disease treatment, which fits in one of the major societal challenges of Europe (Health, Demographic Change and Wellbeing). Potential users of the project outcomes include biologists, pharmacologists and medical doctors, in cooperation with whom the obtained results can be further developed towards real applications. In a long-term perspective, the results of this project can be of use for patients with RNA-related genetic diseases as well as patients suffering from a range of viral and bacterial infections and certain types of cancer.
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