Characterization of fluorescent, unnatural base pairs

Site-specific fluorescent labeling of DNA and RNA molecules is an important tool for investigations in many areas, including analytical and material sciences and medical applications. We report herein an unnatural base pair system between fluorescent base analogs—2-amino-6-(2-thienyl)purine (s) and 2-amino-6-(2-thiazolyl)purine (v), and their fluorophore-linked pairing partners of 2-oxopyridine (y) derivatives—5-(3-amino-1-propynyl)-y linked with 5-carboxyfluorescein (FAM-y) and 5-[3-(6-aminohexanamido)-1-propynyl]-y linked with FAM (FAM-hx-y). These fluorescent nucleoside 5′-triphosphates are enzymatically incorporated into RNA at desired positions by T7 transcription using the unnatural base pair system. The fluorescent s and v bases in nucleic acids are useful for analyzing the local structural dynamics, and their combinations with FAM-y and FAM-hx-y provide a unique fluorescence resonance energy transfer (FRET) system.