Doubly spin-labeled model RNA duplexes prepared using the above-mentioned complementary-addressed SDSL approach were investigated using Q-band (34 GHz) double electron–electron resonance (PELDOR ( 35), also termed DEER), and the obtained distance distributions corresponded well to the expected values. The following steps include the release of aliphatic amino group by hydrolysis of phosphoramide bond in covalent adduct formed and, finally, the selective coupling of spin label to this amino group via acylation with the respective derivative of N-hydroxysuccinimide ester. The key step of this approach is site-directed alkylation of RNA using a specially designed 4-benzylphosphoramide derivative of oligodeoxyribonucleotide, which must be complementary to the only sequence adjacent to the target site. Recently, a versatile approach to SDSL of RNA has been proposed and implemented in model 10-mer RNAs ( 34). Thus, elaboration of approaches for effective introduction of spin labels in desired locations of long natural structured RNAs remains an attractive task. The main disadvantage of the method is generally low yield of ligation, especially for large RNAs, which is likely caused by low extent of formation of correct duplexes between the RNA fragments and the DNA splint ( 33). This method suggested more than two decades ago ( 32) and widely used for RNA labeling critically requires homogeneous 3′-ends of RNAs, which are obtained, as a rule, by T7 transcription. Moreover, for SDSL of long RNA, spin labeling during oligonucleotide synthesis can be combined with enzymatic ligation utilizing DNA splints to bring together 3′- and 5′-termini of RNA fragments and merge them ( 21, 22). Three main approaches underlie all the methods available for SDSL, namely spin labeling during oligonucleotide synthesis, post-synthetic labeling and non-covalent labeling ( 10, 31). To date, a large number of different methods for spin labeling of RNA has been described. Site directed spin labeling (SDSL) of nucleic acids is an integral part of structural studies exploiting EPR spectroscopy, because the specific attachment of a spin label exactly to the target site allows obtaining unambiguous structural information. In this regard, RNAs evoke enormous interest of researchers because these biopolymers are extremely structurally dynamic macromolecules able to generate a wide set of conformations ( 29) and to form a variety of complexes with proteins ( 30). Electron paramagnetic resonance (EPR) spectroscopy is actively used in studies of structure, dynamics and conformational changes of nucleic acids and their complexes ( 1–28).
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