title: Mapping the interactions of selected antibiotics and their Cu2+ complexes with the antigenomic δ ribozyme.
abstract: The interactions of selected antibiotics with the trans-acting antigenomic delta ribozyme were mapped. Ribozyme with two oligonucleotide substrates was used, one uncleavable with deoxycytidine at the cleavage site, mimicking the initial state of ribozyme, and the other with an all-RNA substrate mimicking, after cleavage, the product state. Mapping was performed with a set of RNA structural probing methods: Pb(2+) -induced cleavage, nuclease digestion, and the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) approach. The experimental results combined with molecular modeling revealed different binding sites for neomycin B, amikacin and actinomycin D inside the ribozyme structure. Neomycin B, an aminoglycoside antibiotic, which strongly inhibited the catalytic properties of delta ribozyme, was bound to the pocket formed by the P1 stem, the P1.1 pseudoknot, and the J4/2 junction. Amikacin showed less effective binding to the ribozyme catalytic core, resulting in weak inhibition. Complexes of these aminoglycosides with Cu(2+) ions were bound to the same ribozyme regions, but more effectively, showing lower Kd values. On the other hand, the Cu(2+) complex of the cyclopeptide antibiotic actinonomycin D was preferentially intercalated into the P2 and the P4 double-stranded region, and was three times more potent in ribozyme inhibition than the free antibiotic. In addition, some differences in SHAPE reactivities between the ribozyme forms containing all-RNA and deoxycytidine-modified substrates in the J4/2 region were detected, pointing to different ribozyme conformations before and after the cleavage event.
authors.affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznań, Poland. wrzesinj@ibch.poznan.pl
authors.forename: Jan
authors.initials: J
authors.lastname: Wrzesinski
authors.forename: Leszek
authors.initials: L
authors.lastname: Błaszczyk
authors.forename: Magdalena
authors.initials: M
authors.lastname: Wrońska
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Kasprowicz
authors.forename: Kamila
authors.initials: K
authors.lastname: Stokowa-Sołtys
authors.forename: Justyna
authors.initials: J
authors.lastname: Nagaj
authors.forename: Milena
authors.initials: M
authors.lastname: Szafraniec
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Kulinski
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Jeżowska-Bojczuk
authors.forename: Jerzy
authors.initials: J
authors.lastname: Ciesiołka
mesh_terms: D000583:Amikacin / Q000737:chemistry / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D003198:Computer Simulation; D003300:Copper / Q000378:metabolism; D003609:Dactinomycin / Q000737:chemistry / Q000378:metabolism; D005601:Framycetin / Q000737:chemistry / Q000378:metabolism; D003698:Hepatitis Delta Virus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D016337:RNA, Catalytic; D003609:Dactinomycin; D005601:Framycetin; D003300:Copper; D000583:Amikacin