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

• Published in: Biology methods and protocols 2016-03, Vol.1 (1)
• Main Authors: Jackson, Alexis, Jani, Saumya, Sala, Carol Davies, Soler-Bistué, Alfonso J C, Zorreguieta, Angeles, Tolmasky, Marcelo E
• Format: Article
• Language: English
• Subjects: Methods Manuscript
• ISSN: 2396-8923; 2396-8923
• DOI: 10.1093/biomethods/bpw001

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 BNA , differ in the chemical nature of the bridge. Chimeric oligomers containing LNA or BNA and 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 BNA /DNA chimeric oligonucleotides that share identical nucleotide sequence but with different configurations were carried out using as target the amikacin resistance ' mRNA. LNA/DNA gapmers with 5 and 3/4 LNA residues at the 5'- and 3'-ends, respectively, were the most efficient EGSs while all BNA /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 to cells in culture, an indication of cellular uptake and biological activity.