Diels–Alder Cycloadditions on Synthetic RNA in Mammalian Cells

Abstract

Inverse electron demand Diels-Alder cycloadditions are extremely useful tools for orthogonal labeling of biomolecules such as proteins or small molecules in a cellular context. In-cell labeling of dienophile-modified RNA oligonucleotides using Diels-Alder cycloaddition reactions has not been demonstrated before. In this study we report site-specific labeling of RNA oligonucleotides modified with norbornene derivatives at a predefined sequence position within an RNA sequence in vitro and in mammalian cells using various tetrazine-fluorophore conjugates. The approach could in future be used as a chemical tool for the detection and investigation of RNA functions in cells minimizing the presumed distortion of RNA functions by a large chemical reporter group such as a fluorophore.

Copper‐Free Postsynthetic Labeling of Nucleic Acids by Means of Bioorthogonal Reactions

“…Combining forward and reverse primers leads to triple‐modified double‐stranded DNA in 75 % yield. In vivo experiments with norbornene‐modified RNA building block 51 and with tetrazine‐fluorophore conjugates in mammalian cells showed that the reactivity depends not only on the tetrazine derivatives but also on steric effects, diffusion, and cell permeability 92. This methodology by Kath‐Schorr et al might find use in the future as a chemical‐biological tool for detection and investigation of RNA functions in cells.…”

Modifizierung der 5′‐Kappe eukaryotischer mRNA für Klick‐Reaktionen und zur Beeinflussung der Translationseffizienz in lebenden Zellen

“…[6,8] Bis auf die von Pyka et al [8] beschriebene Tetrazin-Ligation wurde die Klick-Reaktion an sich jedoch immer nach der Fixierung von Zellen durchgeführt. Die intrazelluläre Markierung einer bestimmten Ziel-mRNA, die keine Veränderungen in ihrer Nukleotidsequenz aufweist, würde die direkte Analyse der subzellulären Lokalisation und des Tr ansports dieser mRNAz ue inem bestimmten Zeitpunkt erlauben.…”

“…[8] Wirz eigen zum ersten Mal die Manipulierung einer bestimmten eukaryotischen mRNAi nl ebenden Zellen. [8] Wirz eigen zum ersten Mal die Manipulierung einer bestimmten eukaryotischen mRNAi nl ebenden Zellen.…”

Dual 5′ Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions

“…Moreover, the photo‐induced click reaction between an alkene and an activated tetrazole is also fluorogenic . For RNA labeling, turn‐on tetrazine‐based fluorogenic conjugates have been used in IEDDA reactions for chemically synthesized siRNA strands that have been modified with a norbornene moiety or enzymatically generated RNA oligonucleotides that contain site‐specifically introduced norbornene‐ or cyclopropene‐modified nucleotides.…”