Inhibiting plant micro RNA activity: molecular SPONGE s, target MIMIC s and STTM s all display variable efficacies against target micro RNA s

Summary Elucidation of micro RNA (mi RNA ) function through a loss‐of‐function approach has proven difficult due to extensive genetic redundancy among most plant and animal mi RNA families. Consequently, mi RNA decoy technologies such as target MIMIC s ( MIM s) and short tandem target MIMIC s ( STTM s) in plants or molecular SPONGE s ( SP s) in animals have been developed to generate loss‐of‐function phenotypes by perturbing endogenous mi RNA activity. To test whether SP s can inhibit plant mi RNA activity, synthetic SP transgenes containing multiple mi RNA binding sites targeting different Arabidopsis mi RNA families were generated. Additionally, their silencing efficacies were compared to the corresponding MIM and STTM transgenes via scoring the frequency and severity of phenotypic abnormalities elicited by each transgene. While SP s with wild‐type mi RNA binding sites have no apparent impact, SP s containing mi RNA binding sites with two central mismatches ( cm SP s) can generate strong loss‐of‐function phenotypes. However, their efficacy varied dramatically, from inducing strong loss‐of‐function phenotypes to failing to produce any phenotypic impact. Variability was also observed when MIM s a nd STTM s were compared to cm SP s. While cm SP 165 / 166 and STTM 165/166 showed a stronger efficacy than MIM 165/166 , MIM 159 was stronger than cm SP 159 and STTM 159. Although increasing the number of mi RNA binding sites or strengthening the free energy of the mi RNA binding site interaction can improve decoy efficacy, clearly additional unknown overriding factors are at play. In conclusion, we demonstrate that no one approach guarantees the strongest mi RNA inhibition, but rather distinct mi RNA families respond differently to the various approaches, suggesting that multiple approaches may need to be taken to generate the desired loss‐of‐function outcome.