In Vitro Transcription Networks Based on Hairpin Promoter Switches

In vitro transcription networks are analogs of naturally occurring gene regulatory networks that consist of synthetic DNA templates that are cross-regulated by their own transcripts. This ability to design and execute in vitro transcription networks has allowed bottom-up construction of complex network topologies with predictable dynamic behavior. Here we describe the simplified design of an in vitro transcription network based on single-stranded synthetic DNA hairpin switches that function similar to molecular beacons, via toehold mediated strand displacement. Systematic construction of increasingly larger circuits was achieved by programming interactions between multiple switches through rational sequence design, and the dynamic behavior of networks was accurately predicted using a simple mathematical model. Ultimately, we engineered a cascade of switches that acted as a Boolean complete NAND gate capable of sensing both DNA and RNA inputs. The tools and framework that have been developed makes the execution of in vitro transcription circuits much simpler, which will enable them to more readily serve as testbeds for nucleic acid computations both in vitro and in vivo.