Small regulatory RNAs (sRNAs) in bacteria often act by base pairing to mRNAs. Direct interactions between an sRNA and its target mRNA can be investigated by electrophoretic mobility shift assay. In this assay, regions engaged in base pairing are analyzed by introducing mutations in one of the RNAs that prevent sRNA–mRNA complex formation, followed by the introduction of complementary mutations in its partner RNA that restore base pairing. Here, we describe the design of a mutational strategy used to analyze the base pairing between two CU-rich regions of the sRNA Rli22 and the AG-rich Shine-Dalgarno region of the mRNA oppA in Listeria monocytogenes. The protocol can be employed for mutational studies of base pairing between any sRNA and its mRNA target(s).