TY  - INPR
A1  - Maghami, Mohammad Ghaem
A1  - Scheitl, Carolin P. M.
A1  - Höbartner, Claudia
T1  - Direct in vitro selection of trans-acting ribozymes for posttranscriptional, site-specific, and covalent fluorescent labeling of RNA
T2  - Journal of the American Chemical Society
N2  - General and efficient tools for site-specific fluorescent or bioorthogonal labeling of RNA are in high demand. Here, we report direct in vitro selection, characterization, and application of versatile trans-acting 2'-5' adenylyl transferase ribozymes for covalent and site-specific RNA labeling. The design of our partially structured RNA pool allowed for in vitro evolution of ribozymes that modify a predetermined nucleotide in cis (i.e. intramolecular reaction), and were then easily engineered for applications in trans (i.e. in an intermolecular setup). The resulting ribozymes are readily designed for specific target sites in small and large RNAs and accept a wide variety of N6-modified ATP analogues as small molecule substrates. The most efficient new ribozyme (FH14) shows excellent specificity towards its target sequence also in the context of total cellular RNA.
KW  - covalent and site-specific RNA labeling
KW  - trans-acting 2'-5' adenylyl transferase ribozymes
KW  - in vitro selection from a structured RNA library
KW  - Ribozyme-catalyzed RNA labeling
KW  - intermolecular applications of ribozymes
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-192333
N1  - This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in Journal of the American Chemical Society, copyright © American Chemical Society after peer review. To access the final edited and published work see https://doi.org/10.1021/jacs.9b10531.
ER  - 
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  -