3 research outputs found
Benzoyl-Protected Hydroxylamines for Improved Chemical Synthesis of Oligonucleotides Containing Nitroxide Spin Labels
Post-print (lokagerð höfundar)Oligonucleotides containing nitroxide spin labels, used in biophysical studies of nucleic acids, are frequently prepared by chemical synthesis. However, during the synthesis of spin‐labeled oligonucleotides, the nitroxides are partially reduced to the corresponding amines. Here we report that a benzoylated hydroxylamine can be used as a protected form of the nitroxide to avoid this reduction. The benzoyl group is stable through the oligonucleotide synthesis and is readily removed under standard oligonucleotide deprotection conditions, yielding a hydroxylamine that is oxidized in situ to the nitroxide. This method was used to incorporate the rigid spin labels Ç and Çm into DNA and RNA oligonucleotides, respectively, including a doubly labeled 36‐nucleotide long DNAzyme. Enzymatic digestion of the spin‐labeled oligonucleotides and subsequent HPLC analysis showed that the nitroxides were intact. This protecting group strategy facilitates the high‐yielding synthesis of spin‐labeled DNA and RNA oligonucleotides using the phosphoramidite method.The authors acknowledge financial support by the Icelandic Research Fund (141062-051). The authors thank Dr S. Jonsdottir for assistance with collecting analytical data for structural characterization of new compounds and members of the Sigurdsson research group for helpful discussions.Peer reviewe
Structure guided fluorescence labeling reveals a two-step binding mechanism of neomycin to its RNA aptamer
Publisher's version (útgefin grein)The ability of the cytidine analog Ç m f to act as a position specific reporter of RNA-dynamics was spectroscopically evaluated. Ç m f-labeled single-and double-stranded RNAs differ in their fluorescence lifetimes, quantum yields and anisotropies. These observables were also influenced by the nucleobases flanking Ç m f. This conformation and position specificity allowed to investigate the binding dynamics and mechanism of neomycin to its aptamer N1 by independently incorporating Ç m f at four different positions within the aptamer. Remarkably fast binding kinetics of neomycin binding was observed with stopped-flow measurements, which could be satisfactorily explained with a two-step binding. Conformational selection was identified as the dominant mechanism.Deutsche Forschungsgemeinschaft (DFG) through the Collaborative Research Center (CRC) 902; ‘Molecular Principles of RNA-based Regulation’ sub-projects A7, B14 and Mercator Fellowship. Funding for open access charge: DFG (CRC902); sub-projects A7, B14 and Mercator Fellowship.Peer Reviewe