27 research outputs found
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 C4Ⲡnuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1â˛,H1Ⲡribose 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
Molecular dynamics re-refinement of two different small RNA loop structures using the original NMR data suggest a common structure
Mechanistic studies of an L-proline-catalyzed pyridazine formation involving a DielsâAlder reaction with inverse electron demand
The mechanism of an L-proline-catalyzed pyridazine formation from acetone and aryl-substituted tetrazines via a DielsâAlder 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-DielsâAlder 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
Gordon H. Dixonâs trace in my personal career and the quantic jump experienced in regulatory information
Recognition of 2â˛-O-Methylated 3â˛-End of piRNA by the PAZ Domain of a Piwi Protein
SummaryPiwi proteins are germline-specific Argonautes that associate with small RNAs called Piwi-interacting RNAs (piRNAs), and together with these RNAs are implicated in transposon silencing. The PAZ domain of Argonaute proteins recognizes the 3â˛-end of the RNA, which in the case of piRNAs is invariably modified with a 2â˛-O-methyl group. Here, we present the solution structure of the PAZ domain from the mouse Piwi protein, MIWI, in complex with an 8-mer piRNA mimic. The methyl group is positioned in a hydrophobic cavity made of conserved amino acids from strand β7 and helix Îą3, where it is contacted by the side chain of methionine-382. Our structure is similar to that of Ago-PAZ, but subtle differences illustrate how the PAZ domain has evolved to accommodate distinct 3Ⲡends from a variety of RNA substrates
Solution structure and metal ion binding sites of the human CPEB3 ribozymeâs P4 domain
Three ribozymes are known to occur in humans, the CPEB3 ribozyme, the CoTC ribozyme, and the hammerhead ribozyme. Here, we present the NMR solution structure of a well-conserved motif within the CPEB3 ribozyme, the P4 domain. In addition, we discuss the binding sites and impact of Mg2+ and [Co(NH3)6]3+, a spectroscopic probe for [Mg(H2O)6]2+, on the structure. The well-defined P4 region is a hairpin closed with a UGGU tetraloop that shows a distinct electrostatic surface potential and a characteristic, strongly curved backbone trajectory. The P4 hairpin contains two specific Mg2+ binding sites: one outer-sphere binding site close to the proposed CPEB3 ribozyme active site with potential relevance for maintaining a compact fold of the ribozyme core, and one inner-sphere binding site, probably stabilizing the tetraloop structure. The structure of the tetraloop resembles an RNase III recognition structure, as previously described for an AGUU tetraloop. The detailed knowledge of the P4 domain and its metal ion binding preferences thus brings us closer to understanding the importance of Mg2+ binding for the CPEB3 ribozymeâs fold and function in the cell
Sensitivity enhancement of homonuclear multidimensional NMR correlations for labile sites in proteins, polysaccharides, and nucleic acids
Structure determination of noncanonical RNA motifs guided by šH NMR chemical shifts.
Structured noncoding RNAs underlie fundamental cellular processes, but determining their three-dimensional structures remains challenging. We demonstrate that integrating šH 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 Ă
all-heavy-atom r.m.s. deviation) in 18 cases