Inhibiting The Growth Of Methicillin-Resistant Staphylococcus Aureus In Vitro With Antisense Peptide Nucleic Acid Conjugates Targeting The Ftsz Gene

PART 1 SCREENING OF EFFECTIVE ANTISENSE NUCLEOTIDES TARGETING THE FTSZ GENE OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) COMBINE COMPUTER SOFTWARE AND DOT-BLOT HYBRIDIZATIONObjective:To screen the antisense oligonucleotides targeting the ftsZ mRNA of methicillin-resistant Staphylococcus aureus (MRSA)by computer software by using dot spot hybridization, which bind to the mRNA strongly.Methods:ftsZ mRNA optimal secondary structure regions were predicted by computer program (RNA Structure 5.0) and 10 plausible target sites lacking obvious stable secondary structures,such as stem-loops or hairpins, were selected as antisense oligonucleotide probes. One oligonucleotide from the sense strand sequence was used as the negative control. The full length of ftsZ mRNA transcribed and labeled by digoxigenin-11-uridine-5’-triphosphate, to screen the ftsZ mRNA accessible sites which showed strong binding affinity to the designed antisense oligonucleotides by dot blot hybridization.Results:Only six oligonucleotides exhibited different levels of hybridization signals on the dot blot. Of these, the number 3 probe showed the strongest hybridization signal,whereas the negative control (with a sense sequence) showed no hybridized signal.Conclusion:The antisense oligonucleotides were rapidly and reliably screened with this combination of computer software and dot-blot hybridization.which is a reliable,high-flux and rapid way to screen effective antisense oligonucleotides in vitro, and building a platform for screening of effective antisense oligonucleotides targeting all gene.PART 2 INHIBITING THE GROWTH OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS IN VITRO WITH ANTISENSE PEPTIDE NUCLEIC ACID CONJUGATES TARGETING THE FTSZ GENEObjective:To investigate the inhibition effect of the peptide nucleic acids targeting ftsZ gene on gene expression and growth of Methicillin-resistant Staphylococcus aureus in vitro.Methods:In this study, the synthesis of PNA1 was based on the sequence of the selected oligonucleotides which described in PART1, PNA2 was designed to target the region that includes the translation initiation site and the ribosomal-binding site (Shine-Dalgarno sequence) of the ftsZ gene. Scrambled PNA1 was constructed with mismatches to three bases within the antisense PNA1 sequence. Both of the PNAs were conjugated to the peptide formed peptide-PNA (PPNA),Results:PPNA1 and PPNA2 caused concentration-dependent growth inhibition and had bactericidal effects. The minimal bactericidal concentrations of antisense PPNA1 and PPNA2 were 30μmol/L and 40 μmol/L, respectively, the scrambled PPNA had no effect on bacterial growth, confirming the sequence specificity of the probes. RT-PCR showed that the antisense PPNAs dose-dependently suppressed ftsZ mRNA expression.Conclusion:The PPNA targeted in gene could inhibit the growth of CY-11, this highlights the utility of ftsZ as a promising target for the development of new antisense antibacterial agents to treat MRSA infections.