A Suite of Solid‐State NMR Experiments for RNA Intranucleotide Resonance Assignment in a 21 kDa Protein–RNA Complex

Marchanka, Alexander; Simon, Bernd; Carlomagno, Teresa

doi:10.1002/anie.201304779

Cited by 31 publications

(48 citation statements)

References 56 publications

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“…Three RNAs— IRE (29 nt), a riboswitch (63 nt), and HIV-1 RNA (155 nt)— were used to illustrate the usefulness of this approach. We hope that this methodology will open up new avenues for multidimensional heteronuclear and homonuclear solution and solid-state NMR methods to study the structure and dynamics of large RNA, such as full length riboswitches, which have till now remain unexplored (Cherepanov, Glaubitz, & Schwalbe, 2010; Marchanka, Simon, & Carlomagno, 2013). …”

Section: Discussionmentioning

confidence: 99%

Chemo-Enzymatic Synthesis of Selectively 13C/15N-Labeled RNA for NMR Structural and Dynamics Studies

Alvarado

Longhini

LeBlanc

et al. 2014

Methods in Enzymology

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RNAs are an important class of cellular regulatory elements, and they are well characterized by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy in their folded or bound states. However, the apo or unfolded states are more difficult to characterize by either method. Particularly, effective NMR spectroscopy studies of RNAs in the past were hampered by chemical shift overlap of resonances and associated rapid signal loss due to line broadening for RNAs larger than the median size found in the PDB (∼25 nt); most functional riboswitches are bigger than this median size. Incorporation of selective site-specific 13C/15N-labeled nucleotides into RNAs promises to overcome this NMR size limitation. Unlike previous isotopic enrichment methods such as phosphoramidite, de novo, uniform-labeling and selective biomass approaches, this newer chemical-enzymatic selective method presents a number of advantages for producing labeled nucleotides over these other methods. For example total chemical synthesis of nucleotides, followed by solid-phase synthesis of RNA using phosphoramidite chemistry, while versatile in incorporating isotope labels into RNA at any desired position, faces problems of low yields (<10 %) that drop precipitously for oligonucleotides larger than 50 nt; de novo pyrimidine biosynthesis of NTPs, also a robust technique with modest yields of up to 45%, comes at the cost of using 16 enzymes, expensive substrates, and difficulty in making some needed labeling patterns such as selective labeling of the ribose C1′ and C5′ and the pyrimidine nucleobase C2, C4, C5 or C6; the method of biomass-produced uniformly- or selectively-labeled NTPs suffers from low overall yield per labeled input metabolite and isotopic scrambling with only modest suppression of 13C-13C couplings. In contrast, our current chemo-enzymatic approach overcomes most of these shortcomings and allows for the synthesis of gram quantities of nucleotides with >80% yields while using a limited number of enzymes, six at most. The unavailability of selectively labeled ribose and base precursors had prevented the effective use of this versatile method until now. Recently, we combined an improved organic synthetic approach that selectively places 13C/15N labels in the pyrimidine nucleobase (either 15N1, 15N3, 13C2, 13C4, 13C5, or 13C6 or any combination) with a very efficient enzymatic method to couple ribose with uracil to produce previously unattainable labeling patterns (Alvarado et al 2014). Herein we provide detailed steps of both our chemo-enzymatic synthesis of custom nucleotides and their incorporation into RNAs with sizes ranging from 29 to 155 nt, and showcase the dramatic improvement in spectral quality of reduced crowding and narrow linewidths. Applications of this selective labeling technology should prove valuable in overcoming two major obstacles, chemical shift overlap of resonances and associated rapid signal loss due to line broadening, that have impeded studying the structure and dynamics of large RNAs such as full l...

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Section: Discussionmentioning

confidence: 99%

Chemo-Enzymatic Synthesis of Selectively 13C/15N-Labeled RNA for NMR Structural and Dynamics Studies

Alvarado

Longhini

LeBlanc

et al. 2014

Methods in Enzymology

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“…We note that our approach may also offer advantages for the study of complex materials such as bone [39] or RNAand RNA-protein complexes,which can be challenging for NMR analysis due to the presence of only four different building blocks. [40,41] We also envisage the application of similar experiments to the analysis of chemicalshift anisotropy (CSA) tensors,w here one additional dimension is needed for the CSA recoupling,a nd the CSA dimension requires large spectral widths. [42]…”

Section: Zuschriftenmentioning

confidence: 99%

Bacteriophage Tail‐Tube Assembly Studied by Proton‐Detected 4D Solid‐State NMR

et al. 2017

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Obtaining unambiguous resonance assignments remains a major bottleneck in solid‐state NMR studies of protein structure and dynamics. Particularly for supramolecular assemblies with large subunits (>150 residues), the analysis of crowded spectral data presents a challenge, even if three‐dimensional (3D) spectra are used. Here, we present a proton‐detected 4D solid‐state NMR assignment procedure that is tailored for large assemblies. The key to recording 4D spectra with three indirect carbon or nitrogen dimensions with their inherently large chemical shift dispersion lies in the use of sparse non‐uniform sampling (as low as 2 %). As a proof of principle, we acquired 4D (H)COCANH, (H)CACONH, and (H)CBCANH spectra of the 20 kDa bacteriophage tail‐tube protein gp17.1 in a total time of two and a half weeks. These spectra were sufficient to obtain complete resonance assignments in a straightforward manner without use of previous solution NMR data.

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“…In addition, our labels afford NMR spectra with improved linewidths and signal-to-noise ratios for RNAs ranging in size from 27- to 155-nt. Finally, the use of these labels would (i) enable accurate measurement of residual dipolar couplings, [38] (ii) improve the acquisition of RNA heteronuclear chemical shifts to facilitate rapid identification of noncanonical RNA structures within RNA-ligand/protein interaction interfaces, [39] (iii) be useful for solid-state NMR of RNA, [40,41] and (iv) be easily expanded to deuterate C5, C6 or both positions.…”

mentioning

confidence: 99%

Regio‐Selective Chemical‐Enzymatic Synthesis of Pyrimidine Nucleotides Facilitates RNA Structure and Dynamics Studies

et al. 2014

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Isotope labeling revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs requires site-specific labeling tools to expedite NMR structural and dynamics studies. Using enzymes from the pentose phosphate pathway, we couple chemically synthesized uracil nucleobase with specifically 13C-labeled ribose to synthesize both UTP and CTP with nearly quantitative yields. This chemo-enzymatic method affords a cost-effective preparation of labels that are unattainable by current methods. The methodology generates versatile 13C and 15N labeling patterns which, when employed with relaxation-optimized NMR spectroscopy, effectively mitigates problems of rapid relaxation that result in low resolution and sensitivity. The methodology is demonstrated with RNAs of various sizes, complexity, and importance: the exon splicing silencer 3 (27 nt), iron responsive element (29 nt), Pro-tRNA (76 nt), and HIV-1 core encapsidation signal (155 nt).

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A Suite of Solid‐State NMR Experiments for RNA Intranucleotide Resonance Assignment in a 21 kDa Protein–RNA Complex

Abstract: Intranucleotide resonance of the 26mer box C/D RNA in complex with the L7Ae protein were assigned by solid-state NMR (ssNMR; see picture) spectroscopy. This investigation opens the way for studying RNA in large protein-RNA complexes by ssNMR spectroscopy.

Cited by 31 publications

References 56 publications

Chemo-Enzymatic Synthesis of Selectively 13C/15N-Labeled RNA for NMR Structural and Dynamics Studies

Chemo-Enzymatic Synthesis of Selectively 13C/15N-Labeled RNA for NMR Structural and Dynamics Studies

Bacteriophage Tail‐Tube Assembly Studied by Proton‐Detected 4D Solid‐State NMR

Regio‐Selective Chemical‐Enzymatic Synthesis of Pyrimidine Nucleotides Facilitates RNA Structure and Dynamics Studies

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