Complementary-addressed site-directed spin labeling of long natural RNAs

Abstract

Nanoscale distance measurements by pulse dipolar Electron paramagnetic resonance (EPR) spectroscopy allow new insights into the structure and dynamics of complex biopolymers. EPR detection requires site directed spin labeling (SDSL) of biomolecule(s), which remained challenging for long RNAs up-to-date. Here, we demonstrate that novel complementary-addressed SDSL approach allows efficient spin labeling and following structural EPR studies of long RNAs. We succeeded to spin-label Hepatitis C Virus RNA internal ribosome entry site consisting of ≈330 nucleotides and having a complicated spatial structure. Application of pulsed double electron–electron resonance provided spin–spin distance distribution, which agrees well with the results of molecular dynamics (MD) calculations. Thus, novel SDSL approach in conjunction with EPR and MD allows structural studies of long natural RNAs with nanometer resolution and can be applied to systems of biological and biomedical significance.

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

“…The analysis of RNA structure and dynamics by EPR spectroscopy requires labeling of the samples with paramagnetic species. This is normally achieved by covalent attachment of persistent nitroxide radicals, although noncovalent labeling has become an interesting alternative . Due to the limited stability of nitroxides, postsynthetic attachment has some advantages.…”

Iminoxyl radicalsvs. tert-butylperoxyl radical in competitive oxidative C–O coupling with β-dicarbonyl compounds. Oxime ether formation prevails over Kharasch peroxidation

“…The proposed system is a convenient source of oxime radicals, which have found wide application in various processes of cyclization [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] and C-O coupling recently. 8,39 It should be noted that oxime radicals remain one of the least studied class of N- oxyl radicals, whereas amine-N-oxyl radicals and imide-N-oxyl radicals are used in numerous elds of chemistry, including oxidation methodology, [40][41][42] living radical polymerization, 40,43 spin-labeling, 44,45 organic magnetic materials design. 46 Oximes are difficult substrates for CDC, since under the action of oxidants they give a complex mixtures of products, including dimers of oxime radicals, 47 or ketones.…”

Advances in the synthesis of nitroxide radicals for use in biomolecule spin labelling

“…Among the most impressive is modification of the 332 nucleotide internal ribosome entry site (IRES) of HCV RNA using a templated approach (Scheme 13). 49 Hybridization of a complementary DNA (cDNA) strand 102 to the HCV RNA 103 stimulates transfer of a benzylamine SDSL handle by site-selective modification of the HCV RNA at the adjacent adenine base. Hydrolysis of the P-N linkage on the cDNA strand releases the benzylamine nucleophile, which is then alkylated with Nhydroxy-succinimide ester 104 to afford the spin-labelled RNA 105.…”