13C-direct detected NMR experiments for the sequential J-based resonance assignment of RNA oligonucleotides

We present here a set of 13C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose 13C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4′ nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1′,H1′ ribose signals. The experiments were applied to two RNA hairpin structures. The current set of 13C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, 13C-direct detected NMR methods constitute useful complements to the conventional 1H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs

Mechanistic studies of an L-proline-catalyzed pyridazine formation involving a Diels–Alder reaction with inverse electron demand

The mechanism of an L-proline-catalyzed pyridazine formation from acetone and aryl-substituted tetrazines via a Diels–Alder reaction with inverse electron demand has been studied with NMR and with electrospray ionization mass spectrometry. A catalytic cycle with three intermediates has been proposed. An enamine derived from L-proline and acetone acts as an electron-rich dienophile in a [4 + 2] cycloaddition with the electron-poor tetrazine forming a tetraazabicyclo[2.2.2]octadiene derivative which then eliminates N2 in a retro-Diels–Alder reaction to yield a 4,5-dihydropyridazine species. The reaction was studied in three variants: unmodified, with a charge-tagged substrate, and with a charge-tagged proline catalyst. The charge-tagging technique strongly increases the ESI response of the respective species and therefore enables to capture otherwise undetected reaction components. With the first two reaction variants, only small intensities of intermediates were found, but the temporal progress of reactants and products could be monitored very well. In experiments with the charge-tagged L-proline-derived catalyst, all three intermediates of the proposed catalytic cycle were detected and characterized by collision-induced dissociation (CID) experiments. Some of the CID pathways of intermediates mimic single steps of the proposed catalytic cycle in the gas phase. Thus, the charge-tagged catalyst proved one more time its superior effectiveness for the detection and study of reactive intermediates at low concentrations

Structure determination of noncanonical RNA motifs guided by ¹H NMR chemical shifts.

Structured noncoding RNAs underlie fundamental cellular processes, but determining their three-dimensional structures remains challenging. We demonstrate that integrating ¹H NMR chemical shift data with Rosetta de novo modeling can be used to consistently determine high-resolution RNA structures. On a benchmark set of 23 noncanonical RNA motifs, including 11 'blind' targets, chemical-shift Rosetta for RNA (CS-Rosetta-RNA) recovered experimental structures with high accuracy (0.6-2.0 Å all-heavy-atom r.m.s. deviation) in 18 cases

Evaluation of (15)N-detected H-N correlation experiments on increasingly large RNAs.

Recently, (15)N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a (15)N-TROSY experiment allowed recording of high quality spectra for high molecular weight proteins, even in the absence of deuteration. Here, we demonstrate the applicability of three (15)N-detected H-N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established (15)N-detected BEST-TROSY experiment, which proves to be the most sensitive (15)N-detected H-N correlation experiment, spectra for five RNA molecules ranging in size from 5 to 100 kDa were recorded. These spectra yielded high resolution in the (15)N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the (1)H- than in the (15)N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that (15)N-detected experiments theoretically should benefit from sensitivity and resolution advantages at higher static fields but that the latter is obscured by exchange dynamics within the RNAs