Determinants of aminoglycoside-binding specificity for rRNA by using mass spectrometry
Richard H. Griffey,Steven A. Hofstadler,Kristin A. Sannes-Lowery,David J. Ecker,Stanley T. Crooke +4 more
TLDR
In this paper, high-resolution MS was used to quantitatively identify the noncovalent binding interactions between mixtures of aminoglycosides and multiple RNA targets simultaneously.Abstract:
We have developed methods for studying the interactions between small molecules and RNA and have applied them to characterize the binding of three classes of aminoglycoside antibiotics to ribosomal RNA subdomains. High-resolution MS was used to quantitatively identify the noncovalent binding interactions between mixtures of aminoglycosides and multiple RNA targets simultaneously. Signal overlap among RNA targets was avoided by the addition of neutral mass tags that direct each RNA target to a unique region of the spectrum. In addition to determining binding affinities, the locations of the binding sites on the RNAs were identified from a protection pattern generated by fragmenting the aminoglycoside/RNA complex. Specific complexes were observed for the prokaryotic rRNA A-site subdomain with ribostamycin, paromomycin, and lividomycin, whereas apramycin preferentially formed a complex with the eukaryotic subdomain. We show that differences in binding between paromomycin and ribostamycin can be probed by using an MS–MS protection assay. We have introduced specific base substitutions in the RNA models and have measured their impact on binding affinity and selectivity. The binding of apramycin to the prokaryotic subdomain strongly depends on the identity of position 1408, as evidenced by the selective increase in affinity for an A1408G mutant. An A1409–G1491 mismatch pair in the prokaryotic subdomain enhanced the binding of tobramycin and bekanamycin. These observations demonstrate the power of MS-based methods to provide molecular insights into small molecule/RNA interactions useful in the design of selective new antimicrobial drugs.read more
Citations
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Journal ArticleDOI
Targeting RNA with small molecules
TL;DR: A comparison study of RNA binding in NMR-based and electrospray Ionization Mass Spectrometry-Based methods found that the former showed superior results while the latter showed better results in relation to the latter.
Journal ArticleDOI
Quantitative determination of noncovalent binding interactions using soft ionization mass spectrometry
TL;DR: In this paper, the results of these studies, as well as the methods employed are reviewed, and the possibility to quantitatively measure solution-phase and gas-phase non-covalent interaction strengths by mass spectrometry opens fascinating perspectives for very high sensitivity screening assays as well and for improved fundamental understanding of the nature of non covalent interactions.
Journal ArticleDOI
Antimicrobial resistance in Escherichia coli.
Laurent Poirel,Jean-Yves Madec,Agnese Lupo,Anne-Kathrin Schink,Nicolas Kieffer,Patrice Nordmann,Patrice Nordmann,Stefan Schwarz +7 more
TL;DR: Of note, coselection and persistence of resistances to critically important antimicrobial agents in human medicine also occurs through the massive use of antimicrobialagents in veterinary medicine, such as tetracyclines or sulfonamides, as long as all those determinants are located on the same genetic elements.
Journal ArticleDOI
Targeting RNA with small-molecule drugs: therapeutic promise and chemical challenges.
José M. Gallego,Gabriele Varani +1 more
TL;DR: The elucidation of the structure of the ribosome and other cellular and viral RNA motifs creates the opportunity for discovering new drug-like compounds that inhibit RNA function.
References
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Studying noncovalent protein complexes by electrospray ionization mass spectrometry
TL;DR: Several applications of ESI-MS are discussed, including protein interactions with metal ions and nucleic acids and subunit protein structures (quaternary structure) and mass spectrometry offers advantages in speed and sensitivity.
Journal ArticleDOI
Interaction of antibiotics with functional sites in 16S ribosomal RNA
Danesh Moazed,Harry F. Noller +1 more
TL;DR: Chemical footprinting shows that several classes of antibiotics protect concise sets of highly conserved nucleotides in 16S ribosomal RNA when bound to ribosomes, having strong implications for the mechanism of action of these antibiotics and for the assignment of functions to specific structural features of 16S rRNA.
Journal ArticleDOI
Structure of the A Site of Escherichia coli 16S Ribosomal RNA Complexed with an Aminoglycoside Antibiotic
TL;DR: The structure of the RNA-paromomycin complex explains binding of diverse aminoglycosides to the ribosome, their specific activity against prokaryotic organisms, and various resistance mechanisms, and provides insight into ribosomes function.
Journal ArticleDOI
Interactions of a small RNA with antibiotic and RNA ligands of the 30S subunit
Prakash Purohit,Seth Stern +1 more
TL;DR: It is reported here that an oligo-ribonucleotide analogue of the decoding region interacts with both antibiotic and RNA ligands of the 30S subunit in a manner that correlates with normal subunit function, suggesting that the intimidating structural complexity of the ribosome can be circumvented.
Journal ArticleDOI
Paromomycin binding induces a local conformational change in the A-site of 16 S rRNA.
TL;DR: In this paper, the A-site RNA structure in its free form has been determined using heteronuclear NMR and compared to the structure of the paromomycin-RNA complex.
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