Nature relieso nr eading and synthesizingt he genetic codew ith high fidelity.N ucleic acid building blocks that are orthogonal tot he canonical A-T and G-C base-pairs are therefore uniquely suitable to facilitate position-specific labeling of nucleic acids. Here, we employ the orthogonal kappa-xanthosine-base-pair for in vitro transcription of labeled RNA. We devised an improved synthetic route to obtain the phosphoramidite of the deoxy-version of the kappa nucleoside in solid phase synthesis. From this DNA template, we demonstrate the reliable incorporation of xanthosined uring in vitro transcription. Using NMR spectroscopy,w eshowt hat xanthosine introduces only minor structural changes in an RNA helix. We furthermore synthesized a clickable 7-deaza-xanthosine, which allowst os ite-specifically modify transcribed RNA molecules with fluorophores or other labels.

Genetic code expansion with the orthogonal Watson—Crick‐like base pair kappa and xanthosine enables position‐selective labeling of RNA without disturbing its native structure. Together with a functionalized 7‐deazaxanthosine derivative allowing posttranscriptional modification, the system is a powerful tool to facilitate RNA preparation for biophysical methods including FRET‐ or PELDOR‐based distance measurements. Furthermore, it allows single NMR signal detection in large RNAs for their unambiguous chemical‐shift assignment or interaction studies. More information can be found in the Full Paper by H. Schwalbe, et al. on page 1800.