Back to Search
Start Over
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
- Source :
- Biology Methods and Protocols
- Publication Year :
- 2016
- Publisher :
- Oxford University Press (OUP), 2016.
-
Abstract
- External guide sequences (EGSs) 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 (BNAs) – modified ribonucleotides that contain a bridge at the 2ʹ,4ʹ-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 BNANC 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 BNANC/DNA chimeric oligonucleotides that share identical nucleotide sequence but with different configurations were carried out using as target the amikacin resistance aac(6ʹ)-Ib mRNA. LNA/DNA gapmers with 5 and 3/4 LNA residues at the 5ʹ- and 3ʹ-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, 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 Acinetobacter baumannii cells in culture, an indication of cellular uptake and biological activity.
- Subjects :
- 0301 basic medicine
antibiotic resistance
RNase P
antisense
030106 microbiology
Biology
Rnase P
General Biochemistry, Genetics and Molecular Biology
03 medical and health sciences
chemistry.chemical_compound
Nucleotide
Locked nucleic acid
Methods Manuscript
chemistry.chemical_classification
Nuclease
EGS technology
Acinetobacter
Oligonucleotide
Nucleic acid sequence
Molecular biology
030104 developmental biology
Biochemistry
chemistry
Nucleic acid
biology.protein
aminoglycoside
General Agricultural and Biological Sciences
DNA
Subjects
Details
- ISSN :
- 23968923
- Volume :
- 1
- Database :
- OpenAIRE
- Journal :
- Biology Methods and Protocols
- Accession number :
- edsair.doi.dedup.....6e52c250033b90aa80006e9f8253c298