ABSTRACT
A bio-orthogonal chemistry-based approach for fluorescent labelling of ribosomal RNA is described. It involves an adenosine analogue modified with trans-cyclooctene and masked 5'-phosphate group using aryl phosphoramidate. The incorporation into rRNA has been confirmed using agarose gel electrophoresis, as well as a highly sensitive UHPLC-MS/MS method. Fluorescent labelling of rRNA has been achieved in live HeLa cells via an inverse electron demand Diels-Alder reaction with a tetrazine conjugated to an Oregon Green fluorophore. This communication describes the stepwise approach that led to the development and characterization of the probe. The results demonstrate a new strategy towards development of future fluorescent probes to investigate the biochemistry of nucleic acids.
Subject(s)
Fluorescent Dyes/chemistry , RNA, Ribosomal/chemistry , Chromatography, High Pressure Liquid/methods , Cycloaddition Reaction , HeLa Cells , Humans , Tandem Mass Spectrometry/methodsABSTRACT
A general strategy for purification of oligonucleotides synthesized by solid phase synthesis is described. It is based on a recently developed concept involving a bio-orthogonal inverse electron demand Diels-Alder reaction between trans-cyclooctene and tetrazine, termed 'click-to-release'. The strategy has been applied towards the synthesis and purification of a model hairpin RNA strand, as well as a 34 nt long aptamer.
Subject(s)
Chromatography, High Pressure Liquid/methods , Click Chemistry , RNA/chemical synthesisABSTRACT
This communication describes a general approach for site-specific fluorescence labelling of RNA using a cytidine triphosphate (CTP) analogue derivatized with a trans-cyclooctene group. The analogue was efficiently incorporated into a model RNA strand using in vitro transcription. Bio-orthogonal reaction with fluorescein-labelled tetrazine was utilized to fluorescently tag the synthetic RNA strand.