Advancing Protein Synthesis and Applications with a Recyclable Genomic DNA Hydrogel Platform in Cell-Free Expression Systems

Proteins have the most dynamic entities, performing the diverse role of macromolecules in the body, functional interaction network-based biochemical reactions within a cell or from an organ. Recombinant protein-based therapeutics provide an important breakthrough in biomedical biotechnology. Recent technical advances in cell-free protein synthesis (CFPS) offer several advantages over cell-based expression systems, including easy modification to favor protein folding, increased non-toxicity, and capability for high-throughput strategies due to small reaction volume and short synthesis time. Herein, we fabricated the genomic DNA hydrogel (GD-gel) using the dual single-strand circular plasmids employed in cell-free protein expression synthesis. By complementary rolling complementary circle amplification (cRCA) with multi-primers, the genomic hydrogel exhibits a significantly enhanced productivity yield of protein. This synthesis platform has the potential to develop biotechnological in sensing, therapeutics, and diagnosis.<br/><br/>In our study, inspired by the previous DNA hydrogel using a rolling circle amplification technique, we describe a strategy to synthesize genomic DNA hydrogel (GD-gel) via self-assembly of elongated DNA strands encoding multimeric genomic sequences. To achieve the self-assembly of genomic hydrogel, we applied rolling circle chain amplification (RCCA) with the dual single-stranded circular plasmids. Importantly, GD-gel only consists of numerous tandem repeats of genomic DNA strands by the rolling circle amplification from the plasmid DNA template. This approach allows the genomic DNA itself not only to create a hydrogel structure but also to become a medium for protein expression in GD-gel. Hence, our GD-gel is likely to enable a new route for cell-free protein production via s self-assembly of multimeric genomic DNA.