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- Biochemie und Chemie (8) (remove)
Redirection of miRNA‐argonaute complexes to specific target sites by synthetic adaptor molecules
(2020)
Dysregulation of miRNAs is connected with a multitude of diseases for which antagomirs and miRNA replacement are discussed as therapeutic options. Here, we suggest an alternative concept based on the redirection of RISCs to non‐native target sites. Metabolically stable DNA‐LNA mixmers are used to mediate the binding of RISCs to mRNAs without any direct base complementarity to the presented guide RNA strand. Physical redirection of a dye‐labeled miRNA model and of specific miRNA‐programmed RISC fractions present in HeLa extracts is demonstrated by pull‐down experiments with biotinylated capture oligonucleotides.
Site-specific cleavage of RNAs derived from the PIM1 3′-UTR by a metal-free artificial ribonuclease
(2019)
Oligonucleotide conjugates of tris(2-aminobenzimidazole) have been reported previously to cleave complementary RNA strands with high levels of sequence and site specificity. The RNA substrates used in these studies were oligonucleotides not longer than 29-mers. Here we show that ~150–400-mer model transcripts derived from the 3′-untranslated region of the PIM1 mRNA reacted with rates and specificities comparable to those of short oligonucleotide substrates. The replacement of DNA by DNA/LNA mixmers further increased the cleavage rate. Tris(2-aminobenzimidazoles) were designed to interact with phosphates and phosphate esters. A cell, however, contains large amounts of phosphorylated species that may cause competitive inhibition of RNA cleavage. It is thus important to note that no loss in reaction rates was observed in phosphate buffer. This opens the way to in-cell applications for this type of artificial nuclease. Furthermore, we disclose a new synthetic method giving access to tris(2-aminobenzimidazoles) in multigram amounts.
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.
A mild synthetic method for N-formyl-Met-Leu-Phe-OH (1) is described. After Fmoc solid phase peptide synthesis, on-bead formylation and HPLC purification, more than 30 mg of the fully 13C/15N-labelled tripeptide 1 could be isolated in a typical batch. This peptide can be easily crystallised and is therefore well suited as a standard sample for setting up solid-state NMR experiments.