Evaluation of accessible regions of Escherichia coli fimH mRNA through computational prediction and experimental investigation

Abstract

Background and Objectives: This study aimed to investigate the accessible regions of the fimH mRNA using computational prediction and dot-blot hybridization to increase the effectiveness of antisense anti-virulence therapeutics against Uropatho- genic Escherichia coli.

Materials and Methods: We predicted the secondary structure of the E. coli fimH mRNA using the Sfold and Mfold Web servers and RNA structure 5.5 program. Considering the predicted secondary structure, accessible regions in mRNA of fimH were determined and oligonucleotides complementary to these regions were synthesized and hybridization activity of those oligonucleotides to the fimH Digoxigenin (DIG) labeled mRNA was assessed with dot-blot hybridization.

Results: When searching the fimH gene in the GenBank database, two lengths for this gene was discovered in different strains of E. coli. The difference was related to the nine bases in the first part of the gene utilizing either of two translation initiation sites. Based on the bioinformatics analyses, five regions lacking obvious stable secondary structures were selected in mRNA of fimH. The result of dot-blot hybridization exhibited strongest hybridization signal between the antisense oli- gonucleotide number one and fimH labeled mRNA, whereas hybridization signals were not seen for the negative control.

Conclusion: The results obtained here demonstrate that the region contains start codon of fimH mRNA could act as the potential mRNA target site for anti-fimH antisense therapeutics. It is recommended in the future both of utilizing translation initiation sites be targeted with antisense oligomers compounds.