title: Assessment of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: a methodology for inhibition of expression of antibiotic resistance genes.
abstract: EGSs (external guide sequences) are short antisense oligoribonucleotides that elicit RNase P-mediated cleavage of a target mRNA, which results in inhibition of gene expression. EGS technology is used to inhibit expression of a wide variety of genes, a strategy that may lead to development of novel treatments of numerous diseases, including multidrug-resistant bacterial and viral infections. Successful development of EGS technology depends on finding nucleotide analogs that resist degradation by nucleases present in biological fluids and the environment but still elicit RNase P-mediated degradation when forming a duplex with a target mRNA. Previous results suggested that locked nucleic acids (LNA)/DNA chimeric oligomers have these properties. LNA are now considered the first generation of compounds collectively known as bridged nucleic acids (BNA), modified ribonucleotides that contain a bridge at the 2',4'-position of the ribose. LNA and the second generation BNA, known as BNANC, differ in the chemical nature of the bridge. Chimeric oligomers containing LNA or BNANCand deoxynucleotide monomers in different configurations are nuclease resistant and could be excellent EGS compounds. However, not all configurations may be equally active as EGSs. RNase P cleavage assays comparing LNA/DNA and BNANC/DNA chimeric oligonucleotides that share identical nucleotide sequence but with different configurations were carried out using as target the amikacin resistanceaac(6')-IbmRNA. LNA/DNA gapmers with 5 and 3/4 LNA residues at the 5'- and 3'-ends, respectively, were the most efficient EGSs while all BNANC/DNA gapmers showed very poor activity. When the most efficient LNA/DNA gapmer was covalently bound to a cell penetrating peptide (CPP), the hybrid compound conserved the EGS activity as determined by RNase P cleavage assays and reduced the levels of resistance to amikacin when added toAcinetobacter baumanniicells in culture, an indication of cellular uptake and biological activity.
authors.affiliation: Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA, USA.
authors.forename: Alexis
authors.initials: A
authors.lastname: Jackson
authors.affiliation: Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA, USA.
authors.forename: Saumya
authors.initials: S
authors.lastname: Jani
authors.affiliation: Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA, USA; Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of Buenos Aires, Argentina.
authors.forename: Carol Davies
authors.initials: CD
authors.lastname: Sala
authors.affiliation: Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA, USA; Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of Buenos Aires, Argentina.
authors.forename: Alfonso J C
authors.initials: AJ
authors.lastname: Soler-Bistué
authors.affiliation: Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of Buenos Aires, Argentina.
authors.forename: Angeles
authors.initials: A
authors.lastname: Zorreguieta
authors.affiliation: Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA, USA.
authors.forename: Marcelo E
authors.initials: ME
authors.lastname: Tolmasky