Synthesis and applications of RNAs with position-selective labeling and mosaic composition

Knowledge of the structure and dynamics of RNA molecules is critical to understand their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be significantly enhanced by methods that enable incorporation of modified or labeled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. We have developed a hybrid solid-liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labeling. We demonstrate its utility by successfully preparing various isotope- or fluorescently-labeled versions of the 71-nucleotide aptamer domain of an adenine riboswitch(1) for nuclear magnetic resonance (NMR) spectroscopy or single molecule Förster resonance-energy transfer (smFRET), respectively. Those RNAs include molecules that were selectively isotope-labeled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently-labeled in and near kissing loops. These selectively labeled RNAs have the same fold as those transcribed using conventional methods, but greatly simplified the interpretation of NMR spectra. The single-position isotope-labeled and fluorescently-labeled RNA samples revealed multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labeling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection(2) and disease diagnostics(3,4).