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Dynamic nuclear polarization (DNP) of a biomolecule tagged with a polarizing agent has the potential to not only increase NMR sensitivity but also to provide specificity towards the tagging site. Although the general concept has been often discussed, the observation of true site-specific DNP and its dependence on the electron–nuclear distance has been elusive. Here, we demonstrate site-specific DNP in a uniformly isotope-labeled ubiquitin. By recombinant expression of three different ubiquitin point mutants (F4C, A28C, and G75C) post-translationally modified with a Gd(3+)-chelator tag, localized metal-ion DNP of (13)C and (15)N is investigated. Effects counteracting the site-specificity of DNP such as nuclear spin-lattice relaxation and proton-driven spin diffusion have been attenuated by perdeuteration of the protein. Particularly for (15)N, large DNP enhancement factors on the order of 100 and above as well as localized effects within side-chain resonances differently distributed over the protein are observed. By analyzing the experimental DNP built-up dynamics combined with structural modeling of Gd(3+)-tags in ubiquitin supported by paramagnetic relaxation enhancement (PRE) in solution, we provide, for the first time, quantitative information on the distance dependence of the initial DNP transfer. We show that the direct (15)N DNP transfer rate indeed linearly depends on the square of the hyperfine interaction between the electron and the nucleus following Fermi’s golden rule, however, below a certain distance cutoff paramagnetic signal bleaching may dramatically skew the correlation. Direct DNP transfer rates can be used to measure electron-nuclear distances and to provide site-specificity in NMR.

The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1–D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical β2-β3-β5 interfaces. Evidently, the 3′-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.

NMR spectroscopy is a potent method for the structural and biophysical characterization of RNAs. The application of NMR spectroscopy is restricted in RNA size and most often requires isotope-labeled or even selectively labeled RNAs. Additionally, new NMR pulse sequences, such as the heteronuclear-detected NMR experiments, are introduced. We herein provide detailed protocols for the preparation of isotope-labeled RNA for NMR spectroscopy via in vitro transcription. This protocol covers all steps, from the preparation of DNA template to the transcription of milligram RNA quantities. Moreover, we present a protocol for a chemo-enzymatic approach to introduce a single modified nucleotide at any position of any RNA. Regarding NMR methodology, we share protocols for the implementation of a suite of heteronuclear-detected NMR experiments including 13 C-detected experiments for ribose assignment and amino groups, the CN-spin filter heteronuclear single quantum coherence (HSQC) for imino groups and the 15 N-detected band-selective excitation short transient transverse-relaxation-optimized spectroscopy (BEST-TROSY) experiment. © 2020 The Authors. Basic Protocol 1: Preparation of isotope-labeled RNA samples with in vitro transcription using T7 RNAP, DEAE chromatography, and RP-HPLC purification Alternate Protocol 1: Purification of isotope-labeled RNA from in vitro transcription with preparative PAGE Alternate Protocol 2: Purification of isotope-labeled RNA samples from in vitro transcription via centrifugal concentration Support Protocol 1: Preparation of DNA template from plasmid Support Protocol 2: Preparation of PCR DNA as template Support Protocol 3: Preparation of T7 RNA Polymerase (T7 RNAP) Support Protocol 4: Preparation of yeast inorganic pyrophosphatase (YIPP) Basic Protocol 2: Preparation of site-specific labeled RNAs using a chemo-enzymatic synthesis Support Protocol 5: Synthesis of modified nucleoside 3',5'-bisphosphates Support Protocol 6: Preparation of T4 RNA Ligase 2 Support Protocol 7: Setup of NMR spectrometer for heteronuclear-detected NMR experiments Support Protocol 8: IPAP and DIPAP for homonuclear decoupling Basic Protocol 3: 13 C-detected 3D (H)CC-TOCSY, (H)CPC, and (H)CPC-CCH-TOCSY experiments for ribose assignment Basic Protocol 4: 13 C-detected 2D CN-spin filter HSQC experiment Basic Protocol 5: 13 C-detected C(N)H-HDQC experiment for the detection of amino groups Support Protocol 9: 13 C-detected CN-HSQC experiment for amino groups Basic Protocol 6: 13 C-detected "amino"-NOESY experiment Basic Protocol 7: 15 N-detected BEST-TROSY experiment.