title: Analysis of ribosomal inter-subunit sites as targets for complementary oligonucleotides.
abstract: The bacterial ribosome has many functional ribosomal RNA (rRNA) sites. We have computationally analyzed the rRNA regions involved in the interactions between the 30S and 50S subunits. Various properties of rRNA such as solvent accessibility, opening energy, hydrogen bonding pattern, van der Waals energy, thermodynamic stability were determined. Based on these properties we selected rRNA targets for hybridization with complementary 2'-O-methyl oligoribonucleotides (2'-OMe RNAs). Further, the inhibition efficiencies of the designed ribosome-interfering 2'-OMe RNAs were tested using a β-galactosidase assay in a translation system based on the E. coli extract. Several of the oligonucleotides displayed IC50values below 1 μM, which were in a similar range as those determined for known ribosome inhibitors, tetracycline and pactamycin. The calculated opening and van der Waals stacking energies of the rRNA targets correlated best with the inhibitory efficiencies of 2'-OMe RNAs. Moreover, the binding affinities of several oligonucleotides to both 70S ribosomes and isolated 30S and 50S subunits were measured using a double-filter retention assay. Further, we applied heat-shock chemical transformation to introduce 2'-OMe RNAs to E. coli cells and verify inhibition of bacterial growth. We observed high correlation between IC50in the cell-free extract and bacterial growth inhibition. Overall, the results suggest that the computational analysis of potential rRNA targets within the conformationally dynamic regions of inter-subunit bridges can help design efficient antisense oligomers to probe the ribosome function.
authors.affiliation: Centre of New Technologies, University of Warsaw, Warsaw, 02-097, Poland.
authors.forename: Sapna G
authors.initials: SG
authors.lastname: Thoduka
authors.affiliation: Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznań, Poznań, 61-614, Poland.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Zaleski
authors.affiliation: Centre of New Technologies, University of Warsaw, Warsaw, 02-097, Poland.
authors.forename: Zofia
authors.initials: Z
authors.lastname: Dąbrowska
authors.affiliation: Centre of New Technologies, University of Warsaw, Warsaw, 02-097, Poland.|College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Banacha 2c, Warsaw, 02-097, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Równicki
authors.affiliation: Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznań, Poznań, 61-614, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Stróżecka
authors.affiliation: Centre of New Technologies, University of Warsaw, Warsaw, 02-097, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Górska
authors.affiliation: Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznań, Poznań, 61-614, Poland.
authors.forename: Mikołaj
authors.initials: M
authors.lastname: Olejniczak
authors.affiliation: Centre of New Technologies, University of Warsaw, Warsaw, 02-097, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Trylska
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D017076:Computer-Aided Design; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D010142:Pactamycin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012335:RNA, Ribosomal / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D054681:Ribosome Subunits, Large, Bacterial / Q000737:chemistry / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry / Q000378:metabolism
chemical_list: D009841:Oligonucleotides; D012335:RNA, Ribosomal; D010142:Pactamycin
keywords: 2′-O methyl oligoribonucleotides; bacterial ribosome; computer-aided design; inter-subunit bridges; ribosomal RNA descriptors