Toward Memory in a DNA Brush: Site-Specific Recombination Responsive to Polymer Density, Orientation, and Conformation

[Image: see text] Site-specific recombination is a cellular process for the integration, inversion, and excision of DNA segments that could be tailored for memory transactions in artificial cells. Here, we demonstrate the compartmentalization of cascaded gene expression reactions in a DNA brush, starting from the cell-free synthesis of a unidirectional recombinase that exchanges information between two DNA molecules, leading to gene expression turn-on/turn-off. We show that recombination yield in the DNA brush was responsive to gene composition, density, and orientation, with kinetics faster than in a homogeneous dilute bulk solution reaction. Recombination yield scaled with a power law greater than 1 with respect to the fraction of recombining DNA polymers in a dense brush. The exponent approached either 1 or 2, depending on the intermolecular distance in the brush and the position of the recombination site along the DNA contour length, suggesting that a restricted-reach effect between the recombination sites governs the recombination yield. We further demonstrate the ability to encode the DNA recombinase in the same DNA brush with its substrate constructs, enabling multiple spatially resolved orthogonal recombination transactions within a common reaction volume. Our results highlight the DNA brush as a favorable compartment to study DNA recombination, with unique properties for encoding autonomous memory transactions in DNA-based artificial cells.