PT  - JOURNAL ARTICLE
AU  - Chou, Leo Y.T.
AU  - Shih, William M.
TI  - Cell-free transcriptional regulation via nucleic-acid-based transcription factors
AID  - 10.1101/644021
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 644021
4099  - http://biorxiv.org/content/early/2019/05/20/644021.short
4100  - http://biorxiv.org/content/early/2019/05/20/644021.full
AB  - Cells execute complex transcriptional programs by deploying distinct protein regulatory assemblies that interact with cis-regulatory elements throughout the genome. Using concepts from DNA nanotechnology, we synthetically recapitulated this feature in cell-free gene networks actuated by T7 RNA polymerase (RNAP). Our approach involves engineering nucleic-acid hybridization interactions between a T7 RNAP site-specifically functionalized with single-stranded DNA (ssDNA), templates displaying cis-regulatory ssDNA domains, and auxiliary nucleic-acid assemblies acting as artificial transcription factors (TFs). By relying on nucleic-acid hybridization, de novo regulatory assemblies can be computationally designed to emulate features of protein-based TFs, such as cooperativity and combinatorial binding, while offering unique advantages such as programmability, chemical stability, and scalability. We illustrate the use of nucleic-acid TFs to implement transcriptional logic, cascading, feedback, and multiplexing. This framework will enable rapid prototyping of increasingly complex in vitro genetic devices for applications such as portable diagnostics, bio-analysis, and the design of adaptive materials.