Gene Circuit Compartment on Nanointerface Facilitatating Cascade Gene Expression

Cellular genes that are functionally related to each other are usually confined in specialized subcellular compartments for efficient biochemical reactions. Construction of spatially controlled biosynthetic systems will facilitate the study of biological design principles. Herein, we fabricated a gene circuit compartment by coanchoring two function-related genes on surface of gold nanoparticles and investigated the compartment effect on cascade gene expression in a cell-free system. The gene circuit consisted of a T7 RNA polymerase (T7 RNAP) expression cassette as regulatory gene and a fluorescent protein expression cassette as regulated reporter gene. Both the expression cassettes were attached on a Y-shaped DNA nanostructure whose other two branches were mercapto-modified in order to steadily anchor the gene expression cassettes on the surface of gold nanoparticles. Experimental results demonstrated that both the yield and initial expression rate of the fluorescent reporter protein in the gene circuit compartment system were enhanced compared with those in free gene circuit system. Mechanism investigation revealed that the gene circuit compartment on nanoparticle made the regulatory gene and regulated reporter gene spatially proximal at nanoscale, thus effectively improving the transfer efficiency of the regulatory proteins (T7 RNAP) from regulatory genes to the regulated reporter genes in the compartments, and consequently, the biochemical reaction efficiency was significantly increased. This work not only provided a simplified model for rational molecular programming of genes circuit compartments on nanointerface but also presented implications for the cellular structure-function relationship.