Chemo-enzymatic labeling for rapid assignment of RNA molecules

Abstract

Even though Nuclear Magnetic Resonance (NMR) spectroscopy is one of the few techniques capable of determining atomic resolution structures of RNA, it is constrained by two major problems of chemical shift overlap of resonances and rapid signal loss due to line broadening. Emerging tools to tackle these problems include synthesis of atom specifically labeled or chemically modified nucleotides. Herein we review the synthesis of these nucleotides, the design and production of appropriate RNA samples, and the application and analysis of the NMR experiments that take advantage of these labels.

CCR5 RNA Pseudoknots: Residue and Site‐Specific Labeling correlate Internal Motions with microRNA Binding

“…However,s uch measurements using solution-state NMR are difficult forl arge RNA molecules (> 70 nt;n t = nucleotides) owing to severe spectralo verlap, homonuclear 13 Cs calar couplings, and line broadening. Measurements of RNA dynamic behavior sheds important light on sites that interactw ith their binding partners or cellulars timulators.…”

“…[11] This, however,w as found to be highly sequence restrictive and generated very low yields for large RNA molecules. [8b,e] We report herein the characterization of site-specific motions of a9 6ntCCR5R NA À1P RF pseudoknot element that are correlated to interaction with miR-1224 based on the strategy described above using NMR spectroscopy.W ef irst prepared a key precursor (8,1'-13 C)-modified ATPb yc oupling( 8-13 C)-adenine with (1'-13 C)-riboseu sing chemo-enzymatic synthesis with an excellent yield above 90 %, [13] followed by dephosphorylation with recombinants hrimp alkaline phosphatase to afford (8,1'-13 C)-adenosine ( Figure S1, Supporting Information;c ompound 1 in Scheme 1). [12] Of particularp romise are approaches combining site-specific isotope-labeling,s olid-phase synthesis, and enzymatic ligationi ni nvestigating RNA structure and dynamics.…”

“…Unambiguous assignments of A95C 1 '/H1' was readilya chievedb yo verlaying the ribose C1' HMQC spectrum with that of the CCR5R NA containinga ll seventeena denosine residues prepared from in vitro transcription with atom-specific-labeled (8,1'-13 C)-ATP and unlabeled CTP/GTP/UTP ( Figure 2c). Continued incorporation of 13 Cp robes at different positions of the RNA resulted in identification of ribose C1'/H1' atoms of A76, A79, A85, and A90 (Figure 2d,e)a nd base C8/H8 atoms of these residues ( Figure S3 a, b).…”

“…2018, 24,5462 -5468 www.chemeurj.org beling pattern. [16] The eight well-dispersed 13 Figure S4). [16] The eight well-dispersed 13 Figure S4).…”

“…Upon addition of miR-1224, the ribose H1' chemical shift was barely perturbedf rom 5.86 ppm to 5.83 ppm. The A90-C1' chemical shift of the minor- 13 CCPMG RD profiles of the ligated CCR5R NA at 150 MHz 13 CLarmorfrequency.Red dots represente xperimental data and black dots represent repeated experiments. As imilar result was ob-tained for the A90-C8/H8 chemical shift changes (data not shown here).…”