Untersuchungen zu Struktur und Mechanismus eines Diels-Alder Ribozyms

Das Forschungsgebiet der katalytischen RNA hat sich seit der Entdeckung der natürlichen Ribozyme stark weiterentwickelt. Neben der Erforschung der natürlichen Ribozyme hat sich mit der SELEX-Technik (Systematic Evolution of Ligands by Exponential Enrichment) eine Methode entwickelt, die es ermöglicht, künstliche Ribozyme herzustellen. Damit gelang es, die Bandbreite der durch Ribozyme katalysierbare Reaktionen enorm zu erweitern. Unter anderem konnte ein künstlicher RNA Katalysator generiert werden, der eine C-C-Bindungsknüpfung in einer [4+2]-Cycloaddition, eine sogenannten Diels-Alder Reaktion, katalysiert. Dieser künstliche RNA-Katalysator beschleunigt die Reaktion zwischen Anthracen- und Maleimidderivaten als erstes künstliches Ribozym in einer echten enzymatischen Katalyse. Das Ziel der vorliegenden Arbeit lag in der Aufklärung von Struktur und Mechanismus dieses Diels-Alder Ribozyms. Mit der Synthese verschiedener Maleimidderivate und der Untersuchung derer Substrateigenschaften für das Diels-Alder Ribozym konnte die räumliche Struktur und Beschaffenheit des aktiven Zentrums charakterisiert werden. Die Synthese von photoreaktiven Maleimidsubstraten, und der Einsatz dieser in Photoaffinitätsmarkierungexperimenten erlaubten eine erstmalige genaue Lokalisierung des aktiven Zentrums des Ribozyms. Mit der Herstellung schweratommodifizierter Ribozym-Produkt-Konstrukte für die Lösung des Phasenproblems, konnte ein Beitrag zur Lösung der Kristallstruktur geleistet werden. Die Kristallstrukturen des Ribozym-Produkt-Komplexes und des „freien“ Ribozyms zeigten sich in guter Übereinstimmung mit den zuvor erhaltenen Ergebnissen aus Substratvariationstoleranzuntersuchungen und Photoaffinitätsmarkierungsexperimenten. Aus dem Kontext dieser Ergebnisse konnte ein konkretes Bild der Struktur-Funktionsbeziehung des Diels-Alder Ribozyms entworfen werden. Es konnte gezeigt werden, dass die RNA eine λ-förmige verschlungene Pseudoknoten Struktur mit einer vorgeformten Bindungstasche besitzt. Diese Struktur ermöglicht die Katalyse über „proximity“-Effekte im aktiven Zentrum und durch Stabilisierung des Übergangszustandes. In Untersuchungen der Enantioselektivität des Ribozyms konnte eine Abhängigkeit der Enantioselektivität vom verwendeten Reaktionsformat gezeigt werden. In der intramolekularen Katalyse (in cis-Format) erhält man eine genau umgekehrte Enantioselektivität zu der in intermolekularer Katalyse (in trans-Format) erhaltenen Enantioselektivität. Zur Erklärung dieser außergewöhnlichen stereoselektiven Eigenschaft wurde ein kontrollierter Zugang zum aktiven Zentrum durch eine „Vordertür“ oder eine „Hintertür“ postuliert. Die Existenz eines solchen kontrollierten „Hintertürzugangs“ zum aktiven Zentrum konnte in weiteren Untersuchungen zur Enantioselektivität unter Verwendung unterschiedlicher Ethylenglykollinkerlängen in der intramolekularen Katalyse (in cis-Format) untermauert werden. Auf der Basis dieser Ergebnisse konnte ein Modell zur stereoselektiven Erkennung erstellt werden, welches als „3-Punkt-1- Orientierung“ Modell bezeichnet werden kann

Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag

The micro-viscosity and molecular crowding experienced by specific proteins can regulate their dynamics and function within live cells. Taking advantage of the emerging TMP-tag technology, we present the design, synthesis and application of a hybrid genetic-chemical molecular rotor probe whose fluorescence lifetime can report protein-specific micro-environments in live cells

Protein-specific localization of a rhodamine-based calcium-sensor in living cells

A small synthetic calcium sensor that can be site-specifically coupled to any protein of interest in living cells by utilizing the bio-orthogonal HaloTag labeling strategy.</p

Mandipropamid as a chemical inducer of proximity for in vivo applications

Direct control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins

Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag

The micro-viscosity and molecular crowding experienced by specific proteins can regulate their dynamics and function within live cells. Taking advantage of the emerging TMP-tag technology, we present the design, synthesis and application of a hybrid genetic-chemical molecular rotor probe whose fluorescence lifetime can report protein-specific micro-environments in live cells

Optimizing splinted ligation of highly structured small RNAs

The synthesis of highly structured small RNAs containing nonstandard nucleotides is of high interest for structural and functional investigations. A general approach is the joining, by T4 DNA ligase-mediated splinted ligation, of two or more RNA fragments, each of which may contain its own set of modified nucleotides. The RNA fragments hybridize with a complementary DNA splint to form a ternary ligation-competent-complex (LCC), which is then turned over by the DNA ligase. We studied the formation of the LCC and its precursors using size exclusion chromatography combined with a fluorescence detector. The spatial proximity of two cyanine-dye-labeled RNA fragments in LCCs was detected by monitoring FRET. An observed correlation of LCC formation and ligation yields suggests the use of long splints to stabilize LCCs. Splint oligos of increasing length, which in general appear to reduce the number of different hybridization intermediate species found in a reaction mixture, were applied to the synthesis by T4-DNA-ligation of two highly structured target molecules, one a 73mer tRNA, the other a 49mer synthetic ribozyme. A stable LCC could be isolated and turned over with > 95% ligation efficiency. In conclusion, the use of long splints presents a generally applicable means to overcome the low propensity of highly structured RNAs for hybridization, and thus to significantly improve ligation efficiencies

A dark intermediate in the fluorogenic reaction between tetrazine fluorophores and trans-cyclooctene

Fluorogenic labeling via bioorthogonal tetrazine chemistry has proven to be highly successful in fluorescence microscopy of living cells. To date, trans-cyclooctene (TCO) and bicyclonyne have been found to be the most useful substrates for live-cell labeling owing to their fast labeling kinetics, high biocompatibility, and bioorthogonality. Recent kinetic studies of fluorogenic click reactions with TCO derivatives showed a transient fluorogenic effect but could not explain the reaction sequence and the contributions of different intermediates. More recently, fluorescence quenching by potential intermediates has been investigated, suggesting their occurrence in the reaction sequence. However, in situ studies of the click reaction that directly relate these observations to the known reaction sequence are still missing. In this study, we developed a single-molecule fluorescence detection framework to investigate fluorogenic click reactions. In combination with data from ultra-performance liquid chromatography-tandem mass spectrometry, this explains the transient intensity increase by relating fluorescent intermediates to the known reaction sequence of TCO with fluorogenic tetrazine dyes. More specifically, we confirm that the reaction of TCO with tetrazine rapidly forms a fluorescent 4,5-dihydropyridazine species that slowly tautomerizes to a weakly fluorescent 1,4-dihydropyridazine, explaining the observed drop in fluorescence intensity. On a much slower timescale of hours/days, the fluorescence intensity may be recovered by oxidation of the intermediate to a pyridazine. Our findings are of importance for quantitative applications in fluorescence microscopy and spectroscopy as the achieved peak intensity with TCO depends on the specific experimental settings. They clearly indicate the requirement for more robust benchmarking of click reactions with tetrazine dyes and the need for alternative dienophiles with fast reaction kinetics and stable fluorescence emission to further applications in advanced fluorescence microscopy