Protein-RNA interactions play important biological roles and hence reactive RNA probes for cross-linking with proteins are important tools in their identification and study. To this end, we designed and synthesized 5'-O-triphosphates bearing a reactive squaramate group attached to position 5 of cytidine or position 7 of 7-deazaadenosine and used them as substrates for polymerase synthesis of modified RNA. In vitro transcription with T7 RNA polymerase or primer extension using TGK polymerase was used for synthesis of squaramate-modified RNA probes which underwent covalent bioconjugations with amine-linked fluorophore and lysine-containing peptides and proteins including several viral RNA polymerases or HIV reverse transcriptase. Inhibition of RNA-depending RNA polymerases from Japanese Encephalitis virus was observed through formation of covalent cross-link which was partially identified by MS/MS analysis. Thus, the squaramate-linked NTP analogs are useful building blocks for the synthesis of reactive RNA probes for bioconjugations with primary amines and cross-linking with lysine residues.

Department of Organic Chemistry Faculty of Science Charles University Hlavova 8 CZ 12843 Prague 2 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam 2 CZ 16000 Prague 6 Prague Czech Republic

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Hentze, M. W., Castello, A., Schwarzl, T. & Preiss, T. A brave new world of RNA-binding proteins. Nat. Rev. Mol. Cell Biol.19, 327–341 (2018). PubMed

Nechay, M. & Kleiner, R. E. High-throughput approaches to profile RNA-protein interactions. Curr. Opin. Chem. Biol.54, 37–44 (2020). PubMed PMC

Ramanathan, M., Porter, D. F. & Khavari, P. A. Methods to study RNA-protein interactions. Nat. Methods16, 225–234 (2019). PubMed PMC

Bae, J. W., Kwon, S. C., Na, Y., Kim, V. N. & Kim, J.-S. Chemical RNA digestion enables robust RNA-binding site mapping at single amino acid resolution. Nat. Struct. Mol. Biol.27, 678–682 (2020). PubMed

Willis, M., Hicke, B., Uhienbeck, O., Cech, T. & Koch, T. Photocrosslinking of 5-lodouracil-Substituted RNA and DNA to Proteins. Science262, 1255–1257 (1993). PubMed

Kramer, K. et al. Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins. Nat. Methods11, 1064–1070 (2014). PubMed PMC

Panhale, A. et al. CAPRI enables comparison of evolutionarily conserved RNA interacting regions. Nat. Commun.10, 2682 (2019). PubMed PMC

Luo, H. et al. Photocatalytic Chemical Crosslinking for Profiling RNA-Protein Interactions in Living. Cells Angew. Chem. Int. Ed.61, e202202008 (2022). PubMed

Patton, R. D. et al. Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA. RNA26, 1216–1233 (2020). PubMed PMC

Chiaruttini, C. et al. Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits. Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide. Nucleic Acids Res.23, 7657–7676 (1982). PubMed PMC

Zaman, U. et al. Dithiothreitol (DTT) acts as a specific, UV-inducible cross-linker in elucidation of protein-RNA interactions. Mol. Cell. Proteom.14, 3196–3210 (2015). PubMed PMC

Ivancová, I., Leone, D.-L. & Hocek, M. Reactive modifications of DNA nucleobases for labelling, bioconjugations, and cross-linking. Curr. Opin. Chem. Biol.52, 136–144 (2019). PubMed

Dadová, J. et al. Vinylsulfonamide and acrylamide modification of DNA for cross-linking with proteins. Angew. Chem. Int. Ed.52, 10515–10518 (2013). PubMed

Olszewska, A., Pohl, R., Brázdová, M., Fojta, M. & Hocek, M. Chloroacetamide-linked nucleotides and DNA for cross-linking with peptides and proteins. Bioconjugate Chem.27, 2089–2094 (2016). PubMed

Leone, D.-L. et al. Glyoxal-linked nucleotides and DNA for bioconjugations and crosslinking with arginine-containing peptides and proteins. Chem. Eur. J.28, e202104208 (2022). PubMed

Leone, D., Hubálek, M., Pohl, R., Sýkorová, V. & Hocek, M. 1,3‐diketone‐modified nucleotides and DNA for cross‐linking with arginine‐containing peptides and proteins. Angew. Chem. Int. Ed.133, 17523–17527 (2021). PubMed PMC

Guo, A.-D. et al. Spatiotemporal and global profiling of DNA-protein interactions enables discovery of low-affinity transcription factors. Nat. Chem.15, 803–814 (2023). PubMed

Ivancová, I., Pohl, R., Hubálek, M. & Hocek, M. Squaramate-modified nucleotides and DNA for specific cross-linking with lysine-containing peptides and proteins. Angew. Chem. Int. Ed.58, 13345–13348 (2019). PubMed PMC

Liu, Y. & Santi, D. V. m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts. Proc. Natl Acad. Sci. USA97, 8263–8265 (2000). PubMed PMC

Khoddami, V. & Cairns, B. R. Identification of direct targets and modified bases of RNA cytosine methyltransferases. Nat. Biotechnol.31, 458–464 (2013). PubMed PMC

Dai, W. et al. Activity-based RNA-modifying enzyme probing reveals DUS3L-mediated dihydrouridylation. Nat. Chem. Biol.17, 1178–1187 (2021). PubMed PMC

Fantoni, N. Z., El-Sagheer, A. H. & Brown, T. A Hitchhiker’s guide to click-chemistry with nucleic acids. Chem. Rev.121, 7122–7154 (2021). PubMed

George, J. T. & Srivatsan, S. G. Posttranscriptional chemical labeling of RNA by using bioorthogonal chemistry. Methods120, 28–38 (2017). PubMed

Sawant, A. A. et al. A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA. Nucleic Acids Res.44, e16 (2016). PubMed PMC

Walunj, M. B., Tanpure, A. A. & Srivatsan, S. G. Post-transcriptional labeling by using Suzuki-Miyaura cross-coupling generates functional RNA probes. Nucleic Acids Res.46, e65 (2018). PubMed PMC

Someya, T., Ando, A., Kimoto, M. & Hirao, I. Site-specific labeling of RNA by combining genetic alphabet expansion transcription and copper-free click chemistry. Nucleic Acids Res43, 6665–6676 (2015). PubMed PMC

Eggert, F. & Kath-Schorr, S. A cyclopropene-modified nucleotide for site-specific RNA labeling using genetic alphabet expansion transcription. Chem. Commun.52, 7284–7287 (2016). PubMed

Eggert, F., Kulikov, K., Domnick, C., Leifels, P. & Kath-Schorr, S. Iluminated by foreign letters—strategies for site-specific cyclopropene modification of large functional RNAs via in vitro transcription. Methods120, 17–27 (2017). PubMed

Bornewasser, L., Domnick, C. & Kath-Schorr, S. Stronger together for in-cell translation: natural and unnatural base modified mRNA. Chem. Sci.13, 4753–4761 (2022). PubMed PMC

Mattay, J., Dittmar, M. & Rentmeister, A. Chemoenzymatic strategies for RNA modification and labeling. Curr. Opin. Chem. Biol.63, 46–56 (2021). PubMed

Anhäuser, L., Hüwel, S., Zobel, T. & Rentmeister, A. Multiple covalent fluorescence labeling of eukaryotic mRNA at the poly(A) tail enhances translation and can be performed in living cells. Nucleic Acids Res.47, e42 (2019). PubMed PMC

Hartstock, K. et al. MePMe-seq: antibody-free simultaneous m6A and m5C mapping in mRNA by metabolic propargyl labeling and sequencing. Nat. Commun.14, 7154 (2023). PubMed PMC

van Dülmen, M., Muthmann, N. & Rentmeister, A. Chemo-enzymatic modification of the 5’ cap maintains translation and increases immunogenic properties of mRNA. Angew. Chem. Int. Ed.60, 13280–13286 (2021). PubMed PMC

Brunderová, M., Krömer, M., Vlková, M. & Hocek, M. Chloroacetamide-modified nucleotide and RNA for bioconjugations and cross-linking with RNA-binding. Proteins Angew. Chem. Int. Ed.62, e202213764 (2023). PubMed PMC

Bartas, M., Červeň, J., Guziurová, S., Slychko, K. & Pečinka, P. Amino acid composition in various types of nucleic acid-binding proteins. Int. J. Mol. Sci.22, 922 (2021). PubMed PMC

Hacker, S. et al. Global profiling of lysine reactivity and ligandability in the human proteome. Nat. Chem.9, 1181–1190 (2017). PubMed PMC

Ukmar-Godec, T. et al. Lysine/RNA-interactions drive and regulate biomolecular condensation. Nat. Commun.10, 2909 (2019). PubMed PMC

Vaught, J. D., Dewey, T. & Eaton, B. E. T7 RNA polymerase transcription with 5-position modified UTP derivatives. J. Am. Chem. Soc.126, 11231–11237 (2004). PubMed

Pawar, M. G., Nuthanakanti, A. & Srivatsan, S. G. Heavy atom containing fluorescent ribonucleoside analog probe for the fluorescence detection of RNA-ligand binding. Bioconjug. Chem.24, 1367–1377 (2013). PubMed

Milisavljevič, N., Perlíková, P., Pohl, R. & Hocek, M. Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside triphosphates as substrates. Org. Biomol. Chem.16, 5800–5807 (2018). PubMed

Flamme, M., McKenzie, L. K., Sarac, I. & Hollenstein, M. Chemical methods for the modification of RNA. Methods161, 64–82 (2019). PubMed

Liu, Y. et al. Synthesis and applications of RNAs with position-selective labelling and mosaic composition. Nature522, 368–372 (2015). PubMed PMC

Hertler, J. et al. Synthesis of point-modified mRNA. Nucleic Acids Res.50, e115 (2022). PubMed PMC

Brunderová, M. et al. Expedient production of site specifically nucleobase-labelled or hypermodified RNA with engineered thermophilic DNA polymerases. Nat. Commun.15, 3054 (2024). PubMed PMC

Haslecker, R. et al. Extending the toolbox for RNA biology with SegModTeX: a polymerase-driven method for site-specific and segmental labeling of RNA. Nat. Commun.14, 8422 (2023). PubMed PMC

Yoshikawa, M., Kato, T. & Takenishi, T. A novel method for phosphorylation of nucleosides to 5′-nucleotides. Tetrahedron Lett.8, 5065–5068 (1967). PubMed

Kao, C., Zheng, M. & Rüdisser, S. A simple and efficient method to reduce nontemplated nucleotide addition at the 3 terminus of RNAs transcribed by T7 RNA polymerase. RNA5, 1268–1272 (1999). PubMed PMC

Storer, R. I., Aciro, C. & Jones, L. H. Squaramides: physical properties, synthesis and applications. Chem. Soc. Rev.40, 2330 (2011). PubMed

Pomerantz, R. T. et al. Mechanism of nucleotide misincorporation during transcription due to template-strand misalignment. Mol. Cell24, 245–255 (2006). PubMed PMC

Cozens, C., Pinheiro, V. B., Vaisman, A., Woodgate, R. & Holliger, P. A short adaptive path from DNA to RNA polymerases. Proc. Natl Acad. Sci. USA109, 8067–8072 (2012). PubMed PMC

Malet, H. et al. The flavivirus polymerase as a target for drug discovery. Antivir. Res.80, 23–35 (2008). PubMed

Mishra, A. & Rathore, A. S. RNA dependent RNA polymerase (RdRp) as a drug target for SARS-CoV2. J. Biomol. Struct. Dyn.40, 6039–6051 (2022). PubMed PMC

Cubuk, J. et al. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. Nat. Commun.12, 1–17 (2021). PubMed PMC

Singh, A. K. & Das, K. Insights into HIV-1 reverse transcriptase (RT) inhibition and drug resistance from thirty years of structural studies. Viruses14 (2022). PubMed PMC

Boehmer, P. E. RNA binding and R-loop formation by the herpes simplex virus type-1 single-stranded DNA-binding protein (ICP8). Nucleic Acids Res.32, 4576–4584 (2004). PubMed PMC

Konkolova, E. et al. Remdesivir triphosphate can efficiently inhibit the RNA-dependent RNA polymerase from various flaviviruses. Antivir. Res.182, 104899 (2020). PubMed PMC

Kim, Y. G., Yoo, J. S., Kim, J. H., Kim, C. M. & Oh, J. W. Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase. BMC Mol. Biol.8, 1–12 (2007). PubMed PMC

Selisko, B. et al. Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases. Virology351, 145–158 (2006). PubMed

Lovett, S. T. Encoded errors: mutations and rearrangements mediated by misalignment at repetitive DNA sequences. Mol. Microbiol.52, 1243–1253 (2004). PubMed

Lu, G. & Gong, P. Crystal structure of the full-length japanese encephalitis virus NS5 reveals a conserved methyltransferase-polymerase interface. PLoS Pathog.9 (2013). PubMed PMC

Dejmek, M. et al. Non-nucleotide RNA-dependent RNA polymerase inhibitor that blocks SARS-CoV-2 replication. Viruses13, 1585 (2021). PubMed PMC

Marty, M. T. et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem.87, 4370–4376 (2015). PubMed PMC