Native Top‐Down Mass Spectrometry of TAR RNA in Complexes with a Wild‐Type tat Peptide for Binding Site Mapping

Schneeberger, Eva-Maria; Breuker, Kathrin

doi:10.1002/anie.201610836

Cited by 33 publications

(58 citation statements)

References 36 publications

(36 reference statements)

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“…Thus IRMPD generally cleaves noncovalent before covalent bonds, 102 unless the energy required for noncovalent bond cleavage exceeds that for covalent bond cleavage. 53 , 103 Using energies (25% laser power, 180 ms irradiation time) that were sufficient for covalent bond cleavage in 59% of the (M – 2H + 4Na) 2+ ions from ESI and 27% dissociation of the (M + 2H) + ˙ ions from ECD of (M + 2H) 2+ ions resulted in only ∼15% dissociation of the (M – H + 4Na) 2+ ˙/(M – 2H + 4Na) 2+ ions. Out of the ∼15% dissociation products, 11.1% were losses of NH 3 , H 2 O, and H 3 PO 4 from covalent bond cleavage in (M – H + 4Na) 2+ ˙/(M – 2H + 4Na) 2+ ions, and only 3.9% were c , z ˙ or c ˙, z fragments ( b and y fragments from dissociation of the 7.7% nonradical (M – 2H + 4Na) 2+ ions were not observed).…”

Section: Resultsmentioning

confidence: 99%

Replacing H⁺by Na⁺or K⁺in phosphopeptide anions and cations prevents electron capture dissociation

Schneeberger

Breuker

2018

Chem. Sci.

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Section: Resultsmentioning

confidence: 99%

Replacing H⁺by Na⁺or K⁺in phosphopeptide anions and cations prevents electron capture dissociation

Schneeberger

Breuker

2018

Chem. Sci.

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“…This is quite surprising in view of the high stability of guanidinium–phosphate interactions in gaseous ions and the inherent advantages of native MS, for example, that it does not require stable isotope labeling or crystallization, is not limited by crosslinking reactivity, and uses only relatively small quantities of sample material compared with NMR spectroscopy and X‐ray crystallography. Moreover, a number of laboratories have reported that in the gas phase, the strength of noncovalent bonds between RNA or deoxyribonucleic acid (DNA) and basic ligands can even exceed those of covalent bonds. As a case in point, we have recently shown that the noncovalent bonds between trans‐activation responsive (TAR) RNA and a peptide with the arginine‐rich binding region of the trans‐activator of transcription (tat) protein from HIV‐1 are sufficiently strong to survive phosphodiester backbone cleavage of the RNA by collisionally activated dissociation (CAD), which thus allowed its use to probe tat binding sites of TAR RNA by top‐down MS …”

Section: Introductionmentioning

confidence: 99%

“…The unusual stability of noncovalent bonds in the gas phase has been attributed to strong electrostatic interactions, such as salt bridges, ionic and neutral hydrogen bonds, and charge–dipole interactions, of which salt bridges were thought to provide the highest contribution to stability . In support of this hypothesis, calculations suggest that the interaction energy between guanidine and trifluoroacetic acid, that is, the stabilization achieved when the two neutral molecules are brought from infinite distance to equilibrium distance, is ≈70 kJ mol −1 , whereas that of protonated guanidine and trifluoroacetate, that is, the stabilization achieved when the two oppositely charged ions are brought from infinite distance to equilibrium distance, is ≈500 kJ mol −1 .…”

Section: Introductionmentioning

confidence: 99%

Interactions of Protonated Guanidine and Guanidine Derivatives with Multiply Deprotonated RNA Probed by Electrospray Ionization and Collisionally Activated Dissociation

2017

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Interactions of ribonucleic acid (RNA) with guanidine and guanidine derivatives are important features in RNA–protein and RNA–drug binding. Here we have investigated noncovalently bound complexes of an 8‐nucleotide RNA and six different ligands, all of which have a guanidinium moiety, by using electrospray ionization (ESI) and collisionally activated dissociation (CAD) mass spectrometry (MS). The order of complex stability correlated almost linearly with the number of ligand atoms that can potentially be involved in hydrogen‐bond or salt‐bridge interactions with the RNA, but not with the proton affinity of the ligands. However, ligand dissociation of the complex ions in CAD was generally accompanied by proton transfer from ligand to RNA, which indicated conversion of salt‐bridge into hydrogen‐bond interactions. The relative stabilities and dissociation pathways of [RNA+m L−n H]n− complexes with different stoichiometries (m=1–5) and net charge (n= 2–5) revealed both specific and unspecific ligand binding to the RNA.

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“…10,12,13 Besides, we anticipate a potential use of the large SI labelled RNAs in recently reported mass spectrometric methods to localize protein binding sites, which give valuable information for the 3D structure modelling of large RNP particles. 31,32 We currently also focus on the synthesis of per-deuterated RNA building blocks. The building blocks will be beneficial for NMR but also for SAXS/SANS studies in an integrative structural biology approach, as the chemical RNA synthesis allows full control over segmental deuteration.…”

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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Native Top‐Down Mass Spectrometry of TAR RNA in Complexes with a Wild‐Type tat Peptide for Binding Site Mapping

Cited by 33 publications

References 36 publications

Replacing H⁺by Na⁺or K⁺in phosphopeptide anions and cations prevents electron capture dissociation

Replacing H⁺by Na⁺or K⁺in phosphopeptide anions and cations prevents electron capture dissociation

Interactions of Protonated Guanidine and Guanidine Derivatives with Multiply Deprotonated RNA Probed by Electrospray Ionization and Collisionally Activated Dissociation

Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA

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Native Top‐Down Mass Spectrometry of TAR RNA in Complexes with a Wild‐Type tat Peptide for Binding Site Mapping

Cited by 33 publications

References 36 publications

Replacing H+by Na+or K+in phosphopeptide anions and cations prevents electron capture dissociation

Replacing H+by Na+or K+in phosphopeptide anions and cations prevents electron capture dissociation

Interactions of Protonated Guanidine and Guanidine Derivatives with Multiply Deprotonated RNA Probed by Electrospray Ionization and Collisionally Activated Dissociation

Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA

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Replacing H⁺by Na⁺or K⁺in phosphopeptide anions and cations prevents electron capture dissociation

Replacing H⁺by Na⁺or K⁺in phosphopeptide anions and cations prevents electron capture dissociation