Synthesis of spin-labeled riboswitch RNAs using convertible nucleosides and DNA-catalyzed RNA ligation

Structural Analysis of Protein–RNA Complexes in Solution Using NMR Paramagnetic Relaxation Enhancements

“…As with protein spin labels, the intrinsic flexibility of the spin label when attached to the RNA needs to be carefully considered. RNA spin labeling can be used to analyze the conformation and dynamics of larger RNA molecules, as has been shown with HIV-1 TAR RNA (Wunderlich et al, 2013), riboswitches, and spliceosomal U6 snRNA (Buttner, Javadi-Zarnaghi, & Hobartner, 2014;Buttner, Seikowski, Wawrzyniak, Ochmann, & Hobartner, 2013). Furthermore, the RNA-binding orientation with respect to an RNA-binding protein can be determined (Mackereth et al, 2011), and protein-RNA distances can be obtained and used for structure calculation and validation (Allen, Varani, & Varani, 2001).…”

EPR-aided approach for solution structure determination of large RNAs or protein–RNA complexes

“…However, modified and unmodified RNA fragments can be ligated using enzymatic ligation [27][28][29][30] . To our knowledge, ligation to obtain large RNAs containing two nitroxide spin labels has not been reported yet, with the recent exception of a deoxyribozyme-catalysed ligation, which is limited by strict sequence requirements at the sites of ligation 31 . We investigated two possible strategies using splinted T4 DNA ligation.…”

Combining NMR and EPR to Determine Structures of Large RNAs and Protein–RNA Complexes in Solution

“…While two elegant methods have been introduced by the Hobartner lab performing ligations of smaller RNA fragments using deoxyribozymes (Buttner, Javadi-Zarnaghi, & Hobartner, 2014;Buttner, Seikowski, Wawrzyniak, Ochmann, & Hobartner, 2013), these approaches are currently limited by strong sequence requirements at the sites of ligation.…”