TY - JOUR A1 - Steinmetzger, Christian A1 - Bessi, Irene A1 - Lenz, Ann-Kathrin A1 - Höbartner, Claudia T1 - Structure-fluorescence activation relationships of a large Stokes shift fluorogenic RNA aptamer JF - Nucleic Acids Research N2 - The Chili RNA aptamer is a 52 nt long fluorogen-activating RNA aptamer (FLAP) that confers fluorescence to structurally diverse derivatives of fluorescent protein chromophores. A key feature of Chili is the formation of highly stable complexes with different ligands, which exhibit bright, highly Stokes-shifted fluorescence emission. In this work, we have analyzed the interactions between the Chili RNA and a family of conditionally fluorescent ligands using a variety of spectroscopic, calorimetric and biochemical techniques to reveal key structure - fluorescence activation relationships (SFARs). The ligands under investigation form two categories with emission maxima of ~540 nm or ~590 nm, respectively, and bind with affinities in the nanomolar to low-micromolar range. Isothermal titration calorimetry was used to elucidate the enthalpic and entropic contributions to binding affinity for a cationic ligand that is unique to the Chili aptamer. In addition to fluorescence activation, ligand binding was also observed by NMR spectroscopy, revealing characteristic signals for the formation of a G-quadruplex only upon ligand binding. These data shed light on the molecular features required and responsible for the large Stokes shift and the strong fluorescence enhancement of red and green emitting RNA-chromophore complexes. KW - Chili RNA Aptamer KW - fluorogen-activating RNA aptamer (FLAP) KW - Stokes-shifted fluorescence emission KW - key structure - fluorescence activation relationships (SFARs) KW - ligand binding Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-192340 ER - TY - INPR A1 - Höbartner, Claudia A1 - Steinmetzger, Christian A1 - Palanisamy, Navaneethan A1 - Gore, Kiran R. T1 - A multicolor large Stokes shift fluorogen-activating RNA aptamer with cationic chromophores T2 - Chemistry - A European Journal N2 - Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of their internal 4 hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes-shifted emission from several new green and red HBI analogs that are non-fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functional-ization at the C2 position of the imidazolone, this strategy yielded DMHBO\(^+\), which binds to the Chili aptamer with a low-nanomolar K\(_D\). Because of its highly red-shifted fluorescence emission at 592 nm, the Chili–DMHBO\(^+\) complex is an ideal fluorescence donor for Förster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET-based analytical RNA systems. KW - RNA Aptamer KW - fluorescence KW - large Stokes shift KW - fluorescent protein KW - fluorescent resonance energy transfer Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-174197 N1 - This is the pre-peer reviewed version of the following article: Steinmetzger, C. , Palanisamy, N. , Gore, K. . and Höbartner, C. (2018), A multicolor large Stokes shift fluorogen‐activating RNA aptamer with cationic chromophores. Chem. Eur. J. doi:10.1002/chem.201805882, which has been published in final form at https://doi.org/10.1002/chem.201805882. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. ER - TY - INPR A1 - Sednev, Maksim V. A1 - Mykhailiuk, Volodymyr A1 - Choudhury, Priyanka A1 - Halang, Julia A1 - Sloan, Katherine E. A1 - Bohnsack, Markus T. A1 - Höbartner, Claudia T1 - N\(^6\)-methyladenosine-sensitive RNA-cleaving deoxyribozymes T2 - Angewandte Chemie, International Edition N2 - Deoxyribozymes are synthetic enzymes made of DNA that can catalyze the cleavage or formation of phosphodiester bonds and are useful tools for RNA biochemistry. Here we report new RNA-cleaving deoxyribozymes to interrogate the methylation status of target RNAs, thereby providing an alternative method for the biochemical validation of RNA methylation sites containing N\(^6\)-methyladenosine, which is the most wide-spread and extensively investigated natural RNA modification. Using in vitro selection from random DNA, we developed deoxyribozymes that are sensitive to the presence of N\(^6\)-methyladenosine in RNA near the cleavage site. One class of these DNA enzymes shows faster cleavage of methylated RNA, while others are strongly inhibited by the modified nucleotide. The general applicability of the new deoxyribozymes is demonstrated for several examples of natural RNA sequences, including a lncRNA and a set of C/D box snoRNAs, which have been suggested to contain m\(^6\)A as a regulatory element that influences RNA folding and protein binding. KW - N6-methyladenosine KW - RNA modification KW - deoxyribozymes KW - in vitro selection KW - DNA catalyst Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171753 N1 - This is the pre-peer reviewed version of the following article: M.V. Sednev, V. Mykhailiuk, P. Choudhury, J. Halang, K. E. Sloan, M. T. Bohnsack, C. Höbartner, Angew. Chem. Int. Ed. 2018, 57, 15117-15121, which has been published in final form at https://doi.org/10.1002/anie.201808745. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. ER -