Engineering RNAi circuits

Engineered "computing" biological networks are a generalization of endogenous regulatory pathways. They are intended to generate novel biological responses based on preprogrammed processing of multiple molecular signals. We have recently introduced an approach to constructing complex signal processing networks in mammalian cells using RNA interference (RNAi) as the underlying regulatory mechanism. The approach is modular and the circuits contain sensory, computational, and actuation modules. In the sensory module, various molecular signals are transduced into RNAi-compatible effectors such as small interfering RNA, or microRNA. In the computational module, multiple small RNA (sRNA) effectors converge on the small number of output constructs. Here, we describe experimental methods utilized in circuit construction with the focus on the computational module. We emphasize the steps involved in the design of large sRNA sets required for such circuits.