Non‐Covalent Spin‐Labeling of RNA With Short Hairpins Containing the Rigid Spin Label Çm

Understanding the structure and dynamics of nucleic acids is essential for elucidating their complex biological functions. Electron Paramagnetic Resonance (EPR) spectroscopy is a valuable technique to extract such information. However, its application to nucleic acids requires the incorporation of spin labels, usually nitroxides. Rigid spin labels are more informative EPR probes than flexible ones, since they offer high-precision distance measurements and can provide information about the relative orientation of spin labels. However, the synthesis and incorporation of such rigid labels is nontrivial. Here we describe a strategy in which the rigid spin label Çm is incorporated into a small hairpin, which in turn can be used to label several different oligonucleotides noncovalently through helical stacking. We have studied the noncovalent assembly of spin-labeled RNA hairpins with RNA duplexes by both continuous wave (CW) and pulsed dipolar EPR spectroscopy, specifically pulsed electron-electron double resonance (PELDOR, also called DEER). Our data shows that short complementary overhangs facilitate efficient helical stacking, opening the possibility of using noncovalent labeling with Çm-modified hairpins in conjunction with pulsed dipolar EPR spectroscopy for structural studies of larger RNAs and RNA-protein complexes.