Linear double-stranded DNAs as innovative biological parts to implement genetic circuits in mammalian cells.

Synthetic biology employs engineering principles to redesign biological systems for biomedical or industrial purposes. Innovation and application of original biological parts for genetic circuit construction will significantly facilitate and expedite the development of synthetic biology. Here, we built two- or three-input linear double-stranded DNA (ldsDNA)-based Boolean AND gate genetic circuits in mammalian cells. Bioluminescence imaging revealed the feasibility of ldsDNA-based Boolean AND gate circuits in vivo. Inhibition of DNA-PKcs, a pivotal enzyme in nonhomologous end joining, significantly attenuated the output signals from ldsDNA-based Boolean AND gate circuits. We further showed that ldsDNA with additional terminal random nucleotide(s) could undergo end nucleotide deletion and generate in-frame proteins via the Boolean AND gate response. Additionally, ldsDNAs or plasmids with identical overlapping sequences could also serve as input signals for Boolean AND gate genetic circuits. Our work establishes ldsDNAs as innovative biological parts for building low noise-signal ratio Boolean AND gate circuits with application potential in biomedical engineering fields.