A Cytidine Phosphoramidite with Protected Nitroxide Spin Label: Synthesis of a Full-Length TAR RNA and Investigation by In-Line Probing and EPR Spectroscopy.

EPR studies on RNA are complicated by three major obstacles related to the chemical nature of nitroxide spin labels: Decomposition while oligonucleotides are chemically synthesized, further decay during enzymatic strand ligation, and undetected changes in conformational equilibria due to the steric demand of the label. Herein possible solutions for all three problems are presented: A 2-nitrobenzyloxymethyl protective group for nitroxides that is stable under all conditions of chemical RNA synthesis and can be removed photochemically. By careful selection of ligation sites and splint oligonucleotides, high yields were achieved in the assembly of a full-length HIV-1 TAR RNA labeled with two protected nitroxide groups. PELDOR measurements on spin-labeled TAR in the absence and presence of arginine amide indicated arrest of interhelical motions on ligand binding. Finally, even minor changes in conformation due to the presence of spin labels are detected with high sensitivity by in-line probing.

Phosphoramidite building blocks with protected nitroxides for the synthesis of spin-labeled DNA and RNA.

TEMPO spin labels protected with 2-nitrobenzyloxymethyl groups were attached to the amino residues of three different nucleosides: deoxycytidine, deoxyadenosine, and adenosine. The corresponding phosphoramidites could be incorporated by unmodified standard procedures into four different self-complementary DNA and two RNA oligonucleotides. After photochemical removal of the protective group, elimination of formic aldehyde and spontaneous air oxidation, the nitroxide radicals were regenerated in high yield. The resulting spin-labeled palindromic duplexes could be directly investigated by PELDOR spectroscopy without further purification steps. Spin-spin distances measured by PELDOR correspond well to the values obtained from molecular models.