Positional effect of short interfering RNAs expressing vector targeting human RAB5A

Objectjve: To study the structural effect of short interfering RNAs (siRNAs) expressing vector targeting human RAB5A, find the most effective target site of RAB5A mRNA. Methods: The secondary structure of RAB5A mRNA was predicted using the MFold software. Several siRNAs expressing vectors against different sites of RAB5A mRNA were synthesized and transfected into Anip973. Inhibition of RAB5A gene expression was detected by RT-PCR and Western-blot. Results: siRNAs targeting the most accessible sites inhibited 72% and 85% of RAB5A expression, while those targeting the least accessible sites were completely inactive. Effective targeting site of siRNAs was also found in the untranslating region of RAB5A mRNA. Two mismatched base pairs between siRNA and target sequence resulted in a significant reduction of siRNA activity. Conclusions: The secondary structure of RAB5A mRNA analyzed by the software MFold can be used to find the accessible sites, and the efficiency of siRNAs depends on the accessibility of target sites of RAB5A mRNA.