Iranian

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.

Keywords: Acinetobacter.baumannii, Acyl carrier protein (ACP), computational prediction and dot-blot hybridization

     

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