A few examples of translational activation by antisense small noncoding RNAs (sRNAs) have already been discovered in prokaryotic cells, and all of them are through a sense-antisense interaction at the 5'-untranslated region (5'-UTR) of target mRNAs. Here, we report a novel phenomenon of translational activation of prokaryotic gene expression with trans-acting antisense oligonucleotides targeting the coding region of mRNA. Screening of antisense oligonucleotides complementary to the coding sequences of GFP or ZsGreen identified antisense sequences that activate translation of the mRNAs in a concentration-dependent manner. We also found that the translational activation highly depends on the hybridization positions of the antisense strands. Translation-activating antisense oligonucleotides (TAOs) tended to bind to the 5'-region rather than the 3'-region of the mRNA coding region. RNA folding simulation suggested that TAOs may disrupt the structured elements around the translation initiation region (TIR) by pairing with complementary sequences in the mRNA coding region, resulting in an increase in translation efficiency. Further, we demonstrate that number and position of locked nucleic acid (LNA) bases in the antisense strands govern the tendency of up- or down-regulation. Our findings described here may lead to the discovery of a new class of antisense sRNA and the development of a tool for activating desired gene expression in the future. (C) 2012 Elsevier Inc. All rights reserved.