Polymerase‐Mediated Site‐Specific Incorporation of a Synthetic Fluorescent Isomorphic G Surrogate into RNA. Issue 5 (21st December 2016)

• Record Type: Journal Article
• Authors: Li, Yao; Fin, Andrea; McCoy, Lisa; Tor, Yitzhak
• Is Part Of: Angewandte Chemie international edition. Volume 56:Issue 5(2017)
• Page Start: 1303
• Page End: 1307
• Publication Date: 2016-12-21
• Subjects: fluorescence -- hammerhead ribozyme -- nucleosides -- RNA -- transcription initiation; Chemistry -- Periodicals; 540
• DOI: 10.1002/anie.201609327
• Languages: English
• ISSNs: 1433-7851

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 : An enzyme‐mediated approach is used for the assembly of singly modified RNA constructs in which specific G residues are replaced with th G, an emissive isomorphic G surrogate. By strategic modifications, a mechanistic insight into the ribozyme‐mediated cleavage is gained. Emissive features of the modified nucleoside and responsiveness to environmental changes are used to monitor cleavage in real time by steady‐state fluorescence spectroscopy.