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Genetic code expansion facilitates position-selective modification of nucleic acids and proteins
(2020)
Transcription and translation obey to the genetic code of four nucleobases and 21 amino acids evolved over billions of years. Both these processes have been engineered to facilitate the use of non-natural building blocks in both nucleic acids and proteins, enabling researchers with a decent toolbox for structural and functional analyses. Here, we review the most common approaches for how labeling of both nucleic acids as well as proteins in a site-selective fashion with either modifiable building blocks or spectroscopic probes can be facilitated by genetic code expansion. We emphasize methodological approaches and how these can be adapted for specific modifications, both during as well as after biomolecule synthesis. These modifications can facilitate, for example, a number of different spectroscopic analysis techniques and can under specific circumstances even be used in combination.
Genetic code expansion facilitates position‐selective labeling of rna for biophysical studies
(2019)
Nature relies on reading and synthesizing the genetic code with high fidelity. Nucleic acid building blocks that are orthogonal to the 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 xanthosine during in vitro transcription. Using NMR spectroscopy, we show that xanthosine introduces only minor structural changes in an RNA helix. We furthermore synthesized a clickable 7‐deaza‐xanthosine, which allows to site‐specifically modify transcribed RNA molecules with fluorophores or other labels.