A simplified method for constructing artificial microRNAs based on the osa-MIR528 precursor

Artificial miRNAs (amiRNAs) have been used successfully in various plants to silence endogenous genes or viral RNAs. The method of Schwab et al., widely used to construct amiRNAs, requires four PCRs. We describe a simplified method of constructing amiRNA based on the osa-MIR528 backbone using one PCR step by addition of prolonging sequence to the primers. The length of prolonging sequence needed in the osa-MIR528 precursor was determined. Using this method, we constructed amiRNA targeting the Nicotiana benthamiana UPF1 gene and showed that it functioned in silencing UPF1 expression in leaves when expressed transiently.

► Most of the neighboring sequence of osa-MIR528 precursor outside the stemloop is dispensable for producing miR528.
► Prolonging sequence of 9 nts in osa-MIR528 precursor was required and sufficient for miR528 production.
► Artificial miRNA from osa-MIR528 could be constructed with one PCR step.