Design and Characterization of DNA Strand-Displacement Circuits in Serum-Supplemented Cell Medium

Abstract

The functional stability and lifetimes of synthetic molecular circuits in biological environments are important for long-term, stable sensors or controllers of cell or tissue behavior. DNA-based molecular circuits, in particular DNA strand-displacement circuits, provide simple and effective biocompatible control mechanisms and sensors, but are vulnerable to digestion by nucleases present in living tissues and serum-supplemented cell culture. The stability of double-stranded and single-stranded DNA circuit components in serum-supplemented cell medium and the corresponding effect of nuclease-mediated degradation on circuit performance were characterized to determine the major routes of degradation and DNA strand-displacement circuit failure. Simple circuit design choices, such as the use of 5' toeholds within the DNA complexes used as reactants in the strand-displacement reactions and the termination of single-stranded components with DNA hairpin domains at the 3' termini, significantly increase the functional lifetime of the circuit components in the presence of nucleases. Simulations of multireaction circuits, guided by the experimentally measured operation of single-reaction circuits, enable predictive realization of multilayer and competitive-reaction circuit behavior. Together, these results provide a basic route to increased DNA circuit stability in cell culture environments.

DNA-based communication in populations of synthetic protocells

“…One of the long-standing goals of DNA nanotechnology is to create autonomous molecular machines that can operate in harsh biological environments and provide drug-delivery or diagnostic functionality46. Recent studies have shown that the functionality of simple DSD circuits is severely limited in cell culture medium with 10% foetal bovine serum (FBS) 47. We performed a series of experiments to investigate whether proteinosomes could be used to protect DSD networks from degradation in a biological environment.…”

A Molecular Hero Suit for In Vitro and In Vivo DNA Nanostructures

“…They also were tested against a more aggressive exonuclease, snake‐venom phosphodiesterase, where surprisingly the DPAs showed resistance similar to that of Exo III. In a different vein of native structural modification, Fern and Schulman recently studied the effect of different secondary structures on DNA circuit strands . In Dulbecco's modified Eagle medium media supplemented with 10% FBS and competing ssDNA, a 3′ hairpin domain in the DNA circuit increases lifetime and functionality over a 7 h incubation.…”

“…Supplementing media with MgSO 4 reduces denaturation by low cation availability, and addition of inhibitory agents allows the DNA nanostructures to stealthily evade degradation by nucleases. Supplementing with actin or ssDNA (such as poly‐T or salmon sperm DNA) efficiently out compete the more important DNA nanostructure components for nuclease activity. Heat treatment of serum is an effective method of reducing nuclease activity, but it does not support viable cell culturing .…”

Distributed DNA-based Communication in Populations of Synthetic Protocells

“…One of the long-standing goals of DNA nanotechnology is to create autonomous molecular machines that can operate in harsh biological environments and provide drug-delivery or diagnostic functionality 45 . Recent studies have shown that the functionality of simple DSD circuits is severely limited in cell culture medium with 10% fetal bovine serum (FBS) 46 . We performed a series of experiments to investigate whether proteinosomes could be used to protect DSD networks from degradation in a biological environment.…”