Preparation of Site-Specifically Spin-Labeled RNA by in Vitro Transcription Using an Expanded Genetic Alphabet

Recent advances in pulsed electron paramagnetic resonance (EPR) spectroscopy enable studying structure and folding of nucleic acids. An efficient introduction of spin labels at specific positions within the oligonucleotide sequence is a prerequisite. We here present a step-by-step guide to synthesize long RNA oligonucleotides bearing spin labels at specific positions within the sequence. RNA preparation is achieved enzymatically via in vitro transcription using an expanded genetic alphabet. Highly structured, several hundred nucleotides long RNAs with two nitroxide spin labels at specific positions can be prepared by this method.