Synthesis of (6-<sup>13</sup>C)Pyrimidine Nucleotides as Spin-Labels for RNA Dynamics

Wunderlich, Christoph H.; Spitzer, Romana; Santner, Tobias; Fauster, Katja; Tollinger, Martin; Kreutz, Christoph

doi:10.1021/ja302148g

Cited by 74 publications

(90 citation statements)

References 61 publications

(183 reference statements)

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“…The absence of relaxation dispersion at U23-C6 in TAR-UUCG is inconsistent with a previous study (34) reporting significant relaxation dispersion at U23-C6 in TAR-UUCG using Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments and a TAR sample prepared using solid-phase Offset (Hz) synthesis containing 13 C6-enriched pyrimidines. The exchange parameters obtained for U23-C6 in TAR-UUCG in the previous study (34) are within error with those obtained here at 25°C for TAR containing the WT apical loop (Table S1).…”

Section: Resultscontrasting

confidence: 99%

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Invisible RNA state dynamically couples distant motifs

Lee

Dethoff

Al‐Hashimi

2014

Proc. Natl. Acad. Sci. U.S.A.

104

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Using on-and off-resonance carbon and nitrogen R1ρ NMR relaxation dispersion in concert with mutagenesis and NMR chemical shift fingerprinting, we show that the transactivation response element RNA from the HIV-1 exists in dynamic equilibrium with a transient state that has a lifetime of ∼2 ms and population of ∼0.4%, which simultaneously remodels the structure of a bulge, stem, and apical loop. This is accomplished by a global change in strand register, in which bulge residues pair up with residues in the upper stem, causing a reshuffling of base pairs that propagates to the tip of apical loop, resulting in the creation of three noncanonical base pairs. Our results show that transient states can remodel distant RNA motifs and possibly give rise to mechanisms for rapid long-range communication in RNA that can be harnessed in processes such as cooperative folding and ribonucleoprotein assembly.NMR spectroscopy | dynamics | R1ρ relaxation dispersion | nucleic acids I t is now well-established that RNA sequences do not code for a single static structure, but rather, many conformations that populate energetic minima along a free-energy landscape (1, 2). Cellular inputs, ranging from changes in temperature and pH to the binding of proteins, other RNAs, and ligands, can preferentially stabilize select conformations along the landscape, resulting in dynamic changes in RNA structure that drive the multistep catalytic cycles of ribozymes (3), regulatory activities of riboswitches (4) and other RNA-based switches (5), and the dynamic assembly and disassembly of ribonucleoprotein (RNP) complexes (6).A common mode of RNA dynamics involves rearrangements in secondary structure that can melt or create entire hairpins, and thereby expose or sequester key regulatory elements that are several nucleotides long (1,4,7,8). Such secondary structural transitions entail large kinetic barriers, so they are often catalyzed by RNA-binding proteins (9), ATP-dependent chaperones (10), or otherwise occur by modulating cotranscriptional folding (5, 11). Recently, NMR R1ρ relaxation dispersion experiments (12-15) in concert with mutagenesis (16) have helped uncover more labile RNA secondary structural transitions that can take place without assistance from external cofactors at rates that are 2-4 orders of magnitude faster than larger-scale secondary structural rearrangements. These transitions entail excursions away from the energetically favorable ground state (GS) toward lowpopulated (typically populations <15%) and short-lived (lifetime < milliseconds) species often referred to as "excited states" (ES) (12, 13). These invisible RNA ES feature localized reshuffling of base pairing in and around noncanonical motifs such as bulges, internal loops, and apical loops (16) which can also expose or sequester functionally important residues or promote ATPindependent large-scale changes in secondary structure (14, 15). These faster and more localized changes in secondary structure may meet unique demands in RNA-based regulatory functions (16).Using...

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Section: Resultscontrasting

confidence: 99%

“…The exchange parameters obtained for U23-C6 in TAR-UUCG in the previous study (34) are within error with those obtained here at 25°C for TAR containing the WT apical loop (Table S1). We did not observe relaxation dispersion for U23-C6 in TAR-UUCG when using the same buffer conditions as those used in the CPMG study (34) (Fig. S3).…”

Section: Resultssupporting

confidence: 86%

Invisible RNA state dynamically couples distant motifs

Lee

Dethoff

Al‐Hashimi

2014

Proc. Natl. Acad. Sci. U.S.A.

104

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“…10,12 Thus, the fully chemical SI-labelling protocol can be regarded as an expedient expansion to the settled enzymatic procedures to freely chose the number and positioning of SI-labeled residues into a target RNA. In our hands, however, the standard solid phase synthesis methods are not that well suited to produce larger amounts (450 nmol) and purities higher than 95% for RNAs exceeding 60 nts.…”

Section: -13mentioning

confidence: 99%

Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA

et al. 2017

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We showcase the high potential of the 2 0 -cyanoethoxymethyl (CEM) methodology to synthesize RNAs with naturally occurring modified residues carrying stable isotope (SI) labels for NMR spectro- Solution and solid state nuclear magnetic resonance (NMR) spectroscopy have proven to be highly suitable to address structural and dynamic features of RNA. 1-4 A prerequisite to apply state-of-the-art NMR experiments is the introduction of a stable isotope (SI) labelling pattern using 13 C/ 15 N labelled RNA or DNA precursors. [5][6][7][8] The most wide-spread method uses labelled (2 0 -deoxy)-ribonucleotide triphosphates and enzymes to produce the desired RNA or DNA sequence enriched with 13 C and 15 N nuclei. 1,5 This approach enables to produce sufficient amounts of RNA and DNA for NMR spectroscopic applications. This well-established method allows nucleotide specific labeling by mixing a SI-labeled with unlabeled d/rNTPs. Especially in larger RNAs (460 nt) such nucleotide specific SI-labeling can still lead to significant resonance overlap. That is why, the PLOR (position-selective labelling of RNA) method was recently introduced, which holds the promise to site-specifically label RNA using SI-labelled ribonucleotide triphosphates and T7 RNA polymerase. 9 An alternative method was concurrently developed making use of the synthesis of 2 0 -O-tri-iso-propylsilyloxymethylphosphoramidites and solid phase synthesis. 10-13The approach works well for medium sized RNAs up to 50 nts and the synthetic access to the SI-labelled building blocks is well established.10,12 Thus, the fully chemical SI-labelling protocol can be regarded as an expedient expansion to the settled enzymatic procedures to freely chose the number and positioning of SI-labeled residues into a target RNA. In our hands, however, the standard solid phase synthesis methods are not that well suited to produce larger amounts (450 nmol) and purities higher than 95% for RNAs exceeding 60 nts. Due to this restriction, large RNAs are only accessible via enzymatic ligation strategies using T4 RNA/DNA ligase making extra optimization steps necessary or introducing new problems, such as finding the optimal ligation site or issues regarding up-scaling and yield of the ligation product. 14-16 Thus, an improved synthetic procedure to directly address SI-labelling of larger RNAs (460 nt) at amounts suitable for NMR would be highly desirable. We report the synthesis of SI-labelled RNAs ranging in size between 60 to 80 nts capitalizing on the 2 0 -cyanoethoxymethyl (CEM) RNA synthesis method. 17,18 As these CEM building blocks are not commercially available all phosphoramidites were produced in-house and we further synthesized 13 C-/ 15 N-labelled unmodified and naturally occurring modified RNA phosphoramidites (Fig. 1a and b). In detail, we focused on the synthesis of 8- We used these monomer units to produce SI-labelled RNAs exceeding the size limitation of 60 nucleotides for NMR up to 20 nucleotides. The RNAs reported here were synthesized on a 1.3 mmol scale and on a 1000...

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“…We earlier reported on the synthesis of 6‐ 13 C‐pyrimidine RNA phosphoramidites 3d. The uracil and cytosine nucleobases do not offer an appropriate spin topology due to presence of the H5/H6 scalar coupling ( 3 J H5/H6 ≈8 Hz).…”

mentioning

confidence: 99%

“…This issue can be circumvented by the inclusion of a selective deuteration step to replace the proton at position 5 by deuterium yielding 6‐ 13 C‐5‐ d ‐pyrimidine building blocks with a very small D5/H6 scalar coupling constant ( 3 J D5/H6 ≈1 Hz) making 1 H RD experiments feasible even in the absence of deuterium decoupling (Figure 1 c and d). The synthesis of the 6‐ 13 C‐pyrimidine RNA building blocks was described in detail earlier 3d. The additional deuteration step is now included in the supporting information.…”

mentioning

confidence: 99%

Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy

Juen

Wunderlich²,

Nußbaumer

et al. 2016

Angew Chem Int Ed

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In this work an improved stable isotope labeling protocol for nucleic acids is introduced. The novel building blocks eliminate/minimize homonuclear 13C and 1H scalar couplings thus allowing proton relaxation dispersion (RD) experiments to report accurately on the chemical exchange of nucleic acids. Using site‐specific 2H and 13C labeling, spin topologies are introduced into DNA and RNA that make 1H relaxation dispersion experiments applicable in a straightforward manner. The novel RNA/DNA building blocks were successfully incorporated into two nucleic acids. The A‐site RNA was previously shown to undergo a two site exchange process in the micro‐ to millisecond time regime. Using proton relaxation dispersion experiments the exchange parameters determined earlier could be recapitulated, thus validating the proposed approach. We further investigated the dynamics of the cTAR DNA, a DNA transcript that is involved in the viral replication cycle of HIV‐1. Again, an exchange process could be characterized and quantified. This shows the general applicablility of the novel labeling scheme for 1H RD experiments of nucleic acids.

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Synthesis of (6-¹³C)Pyrimidine Nucleotides as Spin-Labels for RNA Dynamics

Cited by 74 publications

References 61 publications

Invisible RNA state dynamically couples distant motifs

Invisible RNA state dynamically couples distant motifs

Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA

Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy

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Synthesis of (6-13C)Pyrimidine Nucleotides as Spin-Labels for RNA Dynamics

Cited by 74 publications

References 61 publications

Invisible RNA state dynamically couples distant motifs

Invisible RNA state dynamically couples distant motifs

Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA

Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy

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Synthesis of (6-¹³C)Pyrimidine Nucleotides as Spin-Labels for RNA Dynamics