Polymerase‐Mediated Site‐Specific Incorporation of a Synthetic Fluorescent Isomorphic G Surrogate into RNA. (21st December 2016)

• Record Type: Journal Article
• Authors: Li, Yao; Fin, Andrea; McCoy, Lisa; Tor, Yitzhak
• Is Part Of: Angewandte Chemie. Volume 129:Number 5(2017)
• Page Start: 1323
• Page End: 1327
• Publication Date: 2016-12-21
• Subjects: Fluoreszenz -- Hammerhead-Ribozym -- Nucleoside -- RNA -- Transkriptionsinitiierung; Chemistry -- Periodicals; 540
• DOI: 10.1002/ange.201609327
• Languages: English
• ISSNs: 0044-8249

Abstract

Abstract: An enzyme‐mediated approach for the assembly of singly modified RNA constructs in which specific G residues are replaced with th G, an emissive isomorphic G surrogate, is reported. Transcription in the presence of th G and native nucleoside triphosphates enforces initiation with the unnatural analogue, yielding 5′‐end modified transcripts that can be mono‐phosphorylated and ligated to provide longer site‐specifically modified RNA constructs. The scope of this unprecedented enzymatic approach to non‐canonical purine‐containing RNAs is explored via the assembly of several altered hammerhead (HH) ribozymes and a singly modified HH substrate. By strategically modifying key positions, a mechanistic insight into the ribozyme‐mediated cleavage is gained. Additionally, the emissive features of the modified nucleoside and its responsiveness to environmental changes can be used to monitor cleavage in real time by steady state fluorescence spectroscopy. Abstract : Ein enzymvermittelter Ansatz ermöglicht den Aufbau einfach modifizierter RNA‐Konstrukte, in denen spezifische G‐Reste durch th G (ein emittierendes isomorphes G‐Surrogat) ersetzt sind. Strategische Modifikationen geben mechanistische Einblicke in die ribozymvermittelte Spaltung. Die Emissionseigenschaften von th G und seine Antwort auf Umgebungsänderungen wurden genutzt, um die Spaltung in Echtzeit durch Steady‐State‐Fluoreszenzspektroskopie zu verfolgen.