Objective      To screen the effective antisense peptide nucleic acids targeting gyrA gene from multidrug-resistant Acinetobacter baumannii

and to evaluate their antimicrobial effects in vitro.        Methods      Two RNA folding computer programs

Mfold and RNA structure 4.6

were used to predict t

he secondary structure of gyrA mRNA

and then 10 antisense oligonucleotides were designed based on free energy theory. The full length of gyrA mRNA was transcribed in vitro and labeled by digoxigenin-11-uridine-5′-triphosphate. Dot blot hybridization was used to screen the gyrA mRNA accessible sites which showed strong binding affinity to the antisense oligonucleotides. Peptide nucleic acid (PNA) was synthesized based on the sequence of antisense oligonucleotide showing high affinity. Another PNA oligomer containing 6 mismatched nucleotides was used as a negative control. Both the 2 PNAs were conjugated to cell penetrating peptide (CPPs) （KFF）

3

K to form peptide-PNA (PPNA). After the bacterial culture was treated with different concentrations of PPNA

OD

600

and viable cell counts were measured to evaluate the growth inhibitory effect of the antisense oligonucleotide. Reverse transcript (RT)-PCR was applied to evaluate the level of gyrA expression.       Results      Of the 10 antisense oligonucleotides

5 showed binding affinity to gyrA mRNA and one of them showed strong binding affinity. PPNA designed based on the oligonucleotide significantly inhibited the growth of the bacterium and gyrA gene expression at a dose of 5 μmol/L

and exhibited anti-bactericidal effect at a dose of 10 μmol/L. Mismatched PPNA had no effect on the bacterial growth.       Conclusion      Combination of computer-aided prediction with dot blot hybridization is a high-flux and rapid way to screen effective antisense oligonucleotides in vitro. The screened anti-gyrA PPNA exerts significant inhibitory effect on the growth and gene expression in the bacterium in vitro.