Enhanced protein synthesis from immobilized circular DNA <i>via</i> triple-helix formation

Efficient immobilization of DNA on solid surfaces is important for the advancement of electrochemical DNA sensors, biosensors, and bioelectrodes. In this study, we established a new method for immobilizing circular DNA through the formation of triple-helical structures, enabling the high-density and oriented attachment of plasmid DNA to the substrate surface. We engineered biotinylated circular DNA containing a triple-stranded region by hybridizing a biotinylated homopyrimidine third strand with homopurine-homopyrimidine sequences in circular DNA. Subsequently, the modified circular DNA was effectively immobilized on a streptavidin-biotin-functionalized substrate. Using a cell-free protein synthesis system, the yield of <i>Discosoma</i> sp. red (DsRed) fluorescent protein synthesized from immobilized circular DNA was approximately 4.6 times higher than that from immobilized linear DNA. Notably, the immobilized circular DNA template was effectively reused in multiple consecutive rounds of protein synthesis, highlighting its potential for repeated application. Overall, our strategy significantly enhances protein synthesis efficiency and provides a robust platform for the development of high-performance DNA arrays, biosensors, and bioelectrodes.