More than Proton Detection-New Avenues for NMR Spectroscopy of RNA.

Ribonucleic acid oligonucleotides (RNAs) play pivotal roles in cellular function (riboswitches), chemical biology applications (SELEX-derived aptamers), cell biology and biomedical applications (transcriptomics). Furthermore, a growing number of RNA forms (long non-coding RNAs, circular RNAs) but also RNA modifications are identified, showing the ever increasing functional diversity of RNAs. To describe and understand this functional diversity, structural studies of RNA are increasingly important. However, they are often more challenging than protein structural studies as RNAs are substantially more dynamic and their function is often linked to their structural transitions between alternative conformations. NMR is a prime technique to characterize these structural dynamics with atomic resolution. To extend the NMR size limitation and to characterize large RNAs and their complexes above 200 nucleotides, new NMR techniques have been developed. This Minireview reports on the development of NMR methods that utilize detection on low-γ nuclei (heteronuclei like 13 C or 15 N with lower gyromagnetic ratio than 1 H) to obtain unique structural and dynamic information for large RNA molecules in solution. Experiments involve through-bond correlations of nucleobases and the phosphodiester backbone of RNA for chemical shift assignment and make information on hydrogen bonding uniquely accessible. Previously unobservable NMR resonances of amino groups in RNA nucleobases are now detected in experiments involving conformational exchange-resistant double-quantum 1 H coherences, detected by 13 C NMR spectroscopy. Furthermore, 13 C and 15 N chemical shifts provide valuable information on conformations. All the covered aspects point to the advantages of low-γ nuclei detection experiments in RNA.

Chemo-Enzymatic Synthesis of Position-Specifically Modified RNA for Biophysical Studies including Light Control and NMR Spectroscopy.

The investigation of non-coding RNAs requires RNAs containing modifications at every possible position within the oligonucleotide. Here, we present the chemo-enzymatic RNA synthesis containing photoactivatable or 13 C,15 N-labelled nucleosides. All four ribonucleotides containing ortho-nitrophenylethyl (NPE) photocages, photoswitchable azobenzene C-nucleotides and 13 C,15 N-labelled nucleotides were incorporated position-specifically in high yields. We applied this approach for the synthesis of light-inducible 2'dG-sensing riboswitch variants and detected ligand-induced structural reorganization upon irradiation by NMR spectroscopy. This chemo-enzymatic method opens the possibility to incorporate a wide range of modifications at any desired position of RNAs of any lengths beyond the limits of solid-phase synthesis.

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

Recently, 15N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a 15N-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 15N-detected H-N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established 15N-detected BEST-TROSY experiment, which proves to be the most sensitive 15N-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 15N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the 1H- than in the 15N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that 15N-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.

Direct ¹³C-detected NMR experiments for mapping and characterization of hydrogen bonds in RNA.

In RNA secondary structure determination, it is essential to determine whether a nucleotide is base-paired and not. Base-pairing of nucleotides is mediated by hydrogen bonds. The NMR characterization of hydrogen bonds relies on experiments correlating the NMR resonances of exchangeable protons and can be best performed for structured parts of the RNA, where labile hydrogen atoms are protected from solvent exchange. Functionally important regions in RNA, however, frequently reveal increased dynamic disorder which often leads to NMR signals of exchangeable protons that are broadened beyond (1)H detection. Here, we develop (13)C direct detected experiments to observe all nucleotides in RNA irrespective of whether they are involved in hydrogen bonds or not. Exploiting the self-decoupling of scalar couplings due to the exchange process, the hydrogen bonding behavior of the hydrogen bond donor of each individual nucleotide can be determined. Furthermore, the adaption of HNN-COSY experiments for (13)C direct detection allows correlations of donor-acceptor pairs and the localization of hydrogen-bond acceptor nucleotides. The proposed (13)C direct detected experiments therefore provide information about molecular sites not amenable by conventional proton-detected methods. Such information makes the RNA secondary structure determination by NMR more accurate and helps to validate secondary structure predictions based on bioinformatics.

Rapid NMR screening of RNA secondary structure and binding.

Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3' end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields µmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA.

Pausing guides RNA folding to populate transiently stable RNA structures for riboswitch-based transcription regulation.

In bacteria, the regulation of gene expression by cis-acting transcriptional riboswitches located in the 5'-untranslated regions of messenger RNA requires the temporal synchronization of RNA synthesis and ligand binding-dependent conformational refolding. Ligand binding to the aptamer domain of the riboswitch induces premature termination of the mRNA synthesis of ligand-associated genes due to the coupled formation of 3'-structural elements acting as terminators. To date, there has been no high resolution structural description of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA element. Here, we show that for the guanine-sensing xpt-pbuX riboswitch from Bacillus subtilis, the conformation of the full-length transcripts is static: it exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand. We show that only the combined matching of transcription rates and ligand binding enables transcription intermediates to undergo ligand-dependent conformational refolding.