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

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3′ and C5′ carbon positions. Consequently the C1′, C2′ and C4′ positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4′ carbon position, such that the C2′ and C3′ positions suffer from multiplet splitting but the C5′ position remains singlet and the C1′ position shows a small amount of residual C1′–C2′ coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5′ position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4′ carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems

Commissioning of the FORS instruments at the ESO VLT

ABSTRACT FORS (FOcal Reducer/low-dispersion Spectrograph) is an all dioptric focal reducer designed for direct imaging, low-dispersion multi-object spectroscopy, imaging polarimetry and spectropolarimetry of faint objects. Two almost identical copies of the instrument (FORS1 and 2) were built by a consortium of three astronomical institutes (Landessternwarte Heidelberg and the University Observatories of Göttingen and München) under contract and in cooperation with ESO. FORS1 was installed in September 1998 and FORS2 in October 1999 at the Cassegrain foci of the ESO VLT unit telescopes nos.1 and 2. FORS1 is in regular operation since April 1999. Regular observations with FORS2 are scheduled to begin in April 2000. There were two commissioning periods foreseen for each of the instruments to test the performance and reliability under various observing conditions. Extensive tests were done on the electro-mechanical functions, image motion due to flexure, optical quality, instrument software, calibration and in particular on the multi-object spectroscopy. Also a detailed characterization of the instrument's properties in the different observing modes has been carried out. In this paper, the procedures of the tests and the results obtained during the commissioning runs are presented in detail and compared with the specifications

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