RESUMO
In comparison to proteins and protein complexes, the size of RNA amenable to NMR studies is limited despite the development of new isotopic labeling strategies including deuteration and ligation of differentially labeled RNAs. Due to the restricted chemical shift dispersion in only four different nucleotides spectral resolution remains limited in larger RNAs. Labeling RNAs with the NMR-active nucleus (19)F has previously been introduced for small RNAs up to 40 nucleotides (nt). In the presented work, we study the natural occurring RNA aptamer domain of the guanine-sensing riboswitch comprising 73 nucleotides from Bacillus subtilis. The work includes protocols for improved in vitro transcription of 2-fluoroadenosine-5'-triphosphat (2F-ATP) using the mutant P266L of the T7 RNA polymerase. Our NMR analysis shows that the secondary and tertiary structure of the riboswitch is fully maintained and that the specific binding of the cognate ligand hypoxanthine is not impaired by the introduction of the (19)F isotope. The thermal stability of the (19)F-labeled riboswitch is not altered compared to the unmodified sequence, but local base pair stabilities, as measured by hydrogen exchange experiments, are modulated. The characteristic change in the chemical shift of the imino resonances detected in a (1)H,(15)N-HSQC allow the identification of Watson-Crick base paired uridine signals and the (19)F resonances can be used as reporters for tertiary and secondary structure transitions, confirming the potential of (19)F-labeling even for sizeable RNAs in the range of 70 nucleotides.
Assuntos
Adenina/química , Flúor , Ressonância Magnética Nuclear Biomolecular/métodos , RNA/química , Ligantes , Conformação de Ácido NucleicoRESUMO
Because truncation artifacts on magnetic resonance (MR) images may be confused with meniscal tears, measures to suppress them were investigated in a human cadaver knee and prospective and retrospective studies of patients. The artifacts were most prominent when the acquisition matrix was 128 x 256 and the 128-pixel (phase-encoded) axis was in a superoinferior (SI) orientation. An anteroposterior (AP) orientation of the 128-pixel axis or use of a 256 x 256 acquisition matrix reduced the prominence of or nearly eliminated the artifacts. A review of reports of MR imaging and arthroscopic examinations of 83 knees yielded eight menisci that were falsely interpreted at MR imaging as having tears. Retrospective review of the images suggested that the errors were due to truncation artifacts in two cases. Truncation artifacts will cause relatively little difficulty if diagnostic observers are aware of their characteristics and simple steps are taken to minimize their prominence, including acquiring images in 192 x 256 or 256 x 256 matrices or AP rather than SI orientation of the phase-encoded (128-pixel) axis of 128 x 256 matrices.