Background: The aim of present study was to investigate the accessible regions of the
acpP mRNA using computational prediction and dot-blot hybridization to increase the effectiveness of antisense based antibiotics against
A.baumannii.
Methods: We predicated the secondary structure of the
A. baumannii acpP mRNA using the Sfold and Mfold Web servers and RNA structure 5.5 program. Considering the predicted secondary structure, accessible regions in mRNA of
acpP were determined and oligonucleotides complementary to these regions were synthesized and hybridization activity of those oligonucleotides to the
acpP labeled mRNA was assessed with dot-blot hybridization. The whole length of
acpP gene was amplified by PCR using forward primer containing the T7 promoter sequence (genomic DNA of
A.baumannii ATCC19606 was used as a template). Then the
acpP mRNA was transcribed
in vitro in the presence of digoxigenin–UTP and purified PCR product as a template.
Results: Based on the bioinformatics analyses, seven regions lacking obvious stable secondary structures in mRNA of
acpP were selected (they were theoretically accessible to complementary antisense). The result of dot-blot hybridization exhibited hybridization signal between the antisense oligonucleotide number 1 (which targeted the start codon region of
acpP mRNA) and
acpP labeled mRNA, whereas hybridization signals were not seen for all other oligonucleotides (numbers 2 to 7) and the negative control (number 8). These results demonstrated that most of the antisense oligonucleotides were not able to reach the
acpP mRNA (most likely the access prohibited with mRNA secondary structural conformation).
Conclusion: Our study suggests that the region contains start codon of
acpP mRNA as the potential mRNA target site for antisense suppression compares to the other sites of mRNA.