Mapping the interactions of selected antibiotics and their Cu²⁺ complexes with the antigenomic delta 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 K d values. On the other hand, the Cu 2+ complex of the cyclopeptide antibiotic actinonomycin D was preferentially intercalated into the P2 and the P4 doublestranded 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.

The Best Peptidomimetic Strategies to Undercover Antibacterial Peptides

“…Binding Zn(II) to Calcitermin increases the positive charge and facilitate the interactions with negatively charged bacterial membranes [ 251 ]. A similar effect can be observed in zinc(II)/Histidine rich glycoprotein (HRG) abundant in human plasma [ 252 ]. Histatins compose a wide group of short, cationic salivary peptides secreted by the parotid and sub-mandibular salivary gland.…”

Application of a fluorescently labeled trans-acting antigenomic HDV ribozyme to monitor antibiotic–RNA interactions

“…The dsDNA templates for the in vitro transcription of the transacting antigenomic HDV ribozyme were prepared as described. 22 Briey, two DNA oligomers were synthesized: A, 5 0 -GAAAAGTGGCTCTCCCTTAGCCATCCGAGTGCTCGGATGCCC AGGTCGGACCGCGAGGAGGTGGAGATGCCC-3 0 and B, 5 0 -TAA-TACGACTCACTATA GGGCATCTCCACC-3 0 (the complementary sequences are underlined in both oligomers and the letters in italics mark the T7 RNA polymerase promoter). Equimolar amounts of both oligomers A and B were annealed.…”

“…We have previously found that the inhibitory properties of some antibiotics signicantly increase when complexed with Cu 2+ , so the impact of Cu 2+ -antibiotic complexes on ribozyme activity was also analyzed. [21][22][23][24] We were curious to know whether Cu 2+ ions might inuence the application of the uorescence-based assay.…”

“…6,20 We recently characterized a set of antibiotics and their copper(II) complexes that interact with the HDV ribozymes in our laboratory. [21][22][23][24] We and others have found, however, that a precise analysis of the impact of antibiotics on HDV ribozyme catalytic activity is usually difficult. The cleavage reaction is oen very fast, which hampers precise determination of the kinetic parameters of the reaction using manual pipetting.…”

Aminoglycoside antibiotics can inhibit or activate twister ribozyme cleavage

“…Aminoglycoside‐binding sites have been identified in rRNA [7,16], HIV trans‐activating region [17], Rev responsive element [18] and autocatalytic ribozymes [19]. Aminoglycosides can enhance hairpin ribozyme activity in the absence of metal ions [20], and they inhibit hammerhead and hepatitis delta virus ribozyme self‐cleavage through direct interactions with the RNA [21–23]. Small doses of tobramycin induce biofilm formation by targeting a putative cyclic diguanosine monophosphate phosphodiesterase associated with cell surface adhesion in Pseudomonas aeruginosa and in Escherichia coli [24].…”