High‐Resolution Studies of Uniformly <sup>13</sup>C,<sup>15</sup>N‐Labeled RNA by Solid‐State NMR Spectroscopy

Cherepanov, Alexey V.; Glaubitz, Clemens; Schwalbe, Harald

doi:10.1002/anie.200906885

Cited by 39 publications

(29 citation statements)

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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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“…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.…”

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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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“…Recently several labs have demonstrated that MAS NMR can characterize nucleic acids (Cherepanov et al, 2010; Huang et al, 2012) and protein-nucleic acid interactions (Asami et al, 2013; Asami & Reif, 2013; Marchanka et al, 2015; Marchanka et al, 2013) including studies of protein-DNA interactions in intact bacteriophages (Morag et al, 2014; Sergeyev et al, 2011; Yu & Schaefer, 2008). …”

Section: Mas Nmr Of Viral Assemblies and Intact Viral Particlesmentioning

confidence: 99%

Structural biology of supramolecular assemblies by magic-angle spinning NMR spectroscopy

Quinn

Polenova

2017

Quart. Rev. Biophys.

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In recent years, exciting developments in instrument technology and experimental methodology have advanced the field of magic angle spinning (MAS) NMR to new heights. Contemporary MAS NMR yields atomic-level insights into structure and dynamics of an astounding range of biological systems, many of which cannot be studied by other methods. With the advent of fast magic angle spinning, proton detection, and novel pulse sequences, large supramolecular assemblies, such as cytoskeletal proteins and intact viruses, are now accessible for detailed analysis. In this review, we will discuss the current MAS NMR methodologies that enable characterization of complex biomolecular systems and will present examples of applications to several classes of assemblies comprising bacterial and mammalian cytoskeleton as well as HIV-1 and bacteriophage viruses. The body of work reviewed herein is representative of the recent advancements in the field, with respect to the complexity of the systems studied, the quality of the data, and the significance to the biology.

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High‐Resolution Studies of Uniformly ¹³C,¹⁵N‐Labeled RNA by Solid‐State NMR Spectroscopy

Cited by 39 publications

References 31 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

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

Structural biology of supramolecular assemblies by magic-angle spinning NMR spectroscopy

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High‐Resolution Studies of Uniformly 13C,15N‐Labeled RNA by Solid‐State NMR Spectroscopy

Cited by 39 publications

References 31 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

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

Structural biology of supramolecular assemblies by magic-angle spinning NMR spectroscopy

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High‐Resolution Studies of Uniformly ¹³C,¹⁵N‐Labeled RNA by Solid‐State NMR Spectroscopy