Conditional siRNA Production in Human Cell Lysate Via Shape and Sequence Transduction with Small Conditional RNAs

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) enables knockdown of a gene of choice, executing the logical operation: silence gene Y. The fact that the siRNA is constitutively active is a significant limitation, making it difficult to confine knockdown to a specific locus and time. To achieve spatiotemporal control over silencing, we seek to engineer small conditional RNAs (scRNAs) that mediate ʻconditional RNAiʼ corresponding to the logical operation: if gene X is transcribed, silence independent gene Y. By appropriately selecting gene X, knockdown of gene Y could then be restricted in a tissue- and time-specific manner. To implement the logic of conditional RNAi, we recently engineered scRNAs that, upon binding to mRNA ʻdetection targetʼ X in a test tube, perform shape and sequence transduction to form a Dicer substrate targeting independent mRNA ʻsilencing targetʼ Y, with subsequent Dicer processing yielding an siRNA targeting mRNA Y for destruction (Hochrein et al., J Am Chem Soc, 2013). Here, we demonstrate conditional siRNA production in human cell lysate; signal transduction is carried out by a single scRNA in a single step. These results demonstrate that scRNA-mediated shape and sequence transduction can proceed in the presence of cellular proteins and nucleic acids and that Dicer cleaves only the signal transduction product, achieving an important step towards the goal of establishing a new paradigm for programmable conditional regulation based on signal transduction with small conditional RNAs.