@article{SteinmetzgerBessiLenzetal.2019, author = {Steinmetzger, Christian and Bessi, Irene and Lenz, Ann-Kathrin and H{\"o}bartner, Claudia}, title = {Structure-fluorescence activation relationships of a large Stokes shift fluorogenic RNA aptamer}, series = {Nucleic Acids Research}, journal = {Nucleic Acids Research}, doi = {10.1093/nar/gkz1084/5628921}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192340}, pages = {gkz1084}, year = {2019}, abstract = {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shift and the strong fluorescence enhancement of red and green emitting RNA-chromophore complexes.}, language = {en} } @unpublished{NeitzBessiKuperetal.2023, author = {Neitz, Hermann and Bessi, Irene and Kuper, Jochen and Kisker, Caroline and H{\"o}bartner, Claudia}, title = {Programmable DNA interstrand crosslinking by alkene-alkyne [2+2] photocycloaddition}, series = {Journal of the American Chemical Society}, journal = {Journal of the American Chemical Society}, edition = {submitted version}, doi = {10.1021/jacs.3c01611}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-311822}, year = {2023}, abstract = {Covalent crosslinking of DNA strands provides a useful tool for medical, biochemical and DNA nanotechnology applications. Here we present a light-induced interstrand DNA crosslinking reaction using the modified nucleoside 5-phenylethynyl-2'-deoxyuridine (\(^{Phe}\)dU). The crosslinking ability of \(^{Phe}\)dU was programmed by base pairing and by metal ion interaction at the Watson-Crick base pairing site. Rotation to intrahelical positions was favored by hydrophobic stacking and enabled an unexpected photochemical alkene-alkyne [2+2] cycloaddition within the DNA duplex, resulting in efficient formation of a \(^{Phe}\)dU-dimer after short irradiation times of a few seconds. A \(^{Phe}\)dU dimer-containing DNA was shown to efficiently bind a helicase complex, but the covalent crosslink completely prevented DNA unwinding, suggesting possible applications in biochemistry or structural biology.}, language = {en} } @article{NeitzBessiKachleretal.2022, author = {Neitz, Hermann and Bessi, Irene and Kachler, Valentin and Michel, Manuela and H{\"o}bartner, Claudia}, title = {Tailored tolane-perfluorotolane assembly as supramolecular base pair replacement in DNA}, series = {Angewandte Chemie International Edition}, volume = {62}, journal = {Angewandte Chemie International Edition}, number = {1}, doi = {10.1002/anie.202214456}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-312575}, year = {2022}, abstract = {Arene-fluoroarene interactions offer outstanding possibilities for engineering of supramolecular systems, including nucleic acids. Here, we implement the tolane-perfluorotolane interaction as base pair replacement in DNA. Tolane (THH) and perfluorotolane (TFF) moieties were connected to acyclic backbone units, comprising glycol nucleic acid (GNA) or butyl nucleic acid (BuNA) building blocks, that were incorporated via phosphoramidite chemistry at opposite positions in a DNA duplex. Thermodynamic analyses by UV thermal melting revealed a compelling stabilization by THH/TFF heteropairs only when connected to the BuNA backbone, but not with the shorter GNA linker. Detailed NMR studies confirmed the preference of the BuNA backbone for enhanced polar π-stacking. This work defines how orthogonal supramolecular interactions can be tailored by small constitutional changes in the DNA backbone, and it inspires future studies of arene-fluoroarene-programmed assembly of DNA.}, language = {en} } @article{MieczkowskiSteinmetzgerBessietal.2021, author = {Mieczkowski, Mateusz and Steinmetzger, Christian and Bessi, Irene and Lenz, Ann-Kathrin and Schmiedel, Alexander and Holzapfel, Marco and Lambert, Christoph and Pena, Vladimir and H{\"o}bartner, Claudia}, title = {Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine}, series = {Nature Communications}, volume = {12}, journal = {Nature Communications}, doi = {10.1038/s41467-021-23932-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254527}, pages = {3549}, year = {2021}, abstract = {Fluorogenic RNA aptamers are synthetic functional RNAs that specifically bind and activate conditional fluorophores. The Chili RNA aptamer mimics large Stokes shift fluorescent proteins and exhibits high affinity for 3,5-dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI) derivatives to elicit green or red fluorescence emission. Here, we elucidate the structural and mechanistic basis of fluorescence activation by crystallography and time-resolved optical spectroscopy. Two co-crystal structures of the Chili RNA with positively charged DMHBO+ and DMHBI+ ligands revealed a G-quadruplex and a trans-sugar-sugar edge G:G base pair that immobilize the ligand by π-π stacking. A Watson-Crick G:C base pair in the fluorophore binding site establishes a short hydrogen bond between the N7 of guanine and the phenolic OH of the ligand. Ultrafast excited state proton transfer (ESPT) from the neutral chromophore to the RNA was found with a time constant of 130 fs and revealed the mode of action of the large Stokes shift fluorogenic RNA aptamer.}, language = {en} }