Generation of DNA-based artificial ion channels and molecular switches toward construction of synthetic cells

Abstract

Bottom-up construction of artificial cells, which are supramolecular chemical systems to mimic essential behaviors, functions, and structures of living cells, is an emerging research field that attempts to build the cells from non-living matters. In this approach, various functional and structural modules are first generated and then integrated into sophisticated systems to reproduce the organizations and functions of living cells, using a wide range of tools in materials science and biochemistry. In this dissertation, I aim to develop several critical building blocks for synthetic cell-like systems, mainly based on programmable DNA nanotechnology, to provide diverse functionalities and lipid membrane systems to realize efficient compartmentalization. When new cytomimetic biomolecular materials are developed to enable 1) selective ion transport and 2) stimulus-triggered regulation of bio- polymerization, they are utilized for diverse bio-applications ranging from biosensors to therapeutics. First, by mimicking biological ion channels, I developed ‘DNA-based artificial ion channels’ for selective ion transport across a self-assembled lipid bilayer. As an example, I demonstrated de novo synthesis of a K+ channel-based selective membrane and its integration with a polyelectrolyte hydrogel-based ionic transistor to achieve real-time K+-selective ion-to-ion current amplification against complex bioenvironments. In-line K+-binding G-quartets were prepared across the freestanding lipid bilayer by G-specific hexyl modifications of monolithic G-quadruplexes, and the pre-filtered K+ flow was directly interpreted into amplified ionic currents by the hydrogel ionic transistor with a fast response time at 100 ms intervals. Through the synergistic combination of ion recognition, charge repulsion, and sieving, the synthetic membrane permitted exclusive K+ transport against other ions without water leakage. K+ was 250 and 17 times more permeable than the monovalent anion, Cl-, and the polyatomic cation, N-methyl-D-glucamine+, respectively, and the molecular recognition-based ion channeling allowed a 500% greater signal of K+ compared to Li+, despite the radius of K+ being 170% larger than that of Li+ of the same valence. The miniaturized device enabled non-invasive, direct, and real-time monitoring of K+ efflux from living cell spheroids with minimal crosstalk, allowing to recording of osmotic shock-induced necrosis and drug-antidote dynamics. The uniqueness of the artificial channel platform devised in this study lies in its ability to expand the target ions that can be selectively filtered out, by simply replacing the ion recognition motifs inserted into the lipid membrane. Indeed, I have additionally generated a DNA-based artificial Ag+ channel by vertically aligning Ag+ binding motifs in a row within the lipid membranes, which is an unprecedented system in nature. Furthermore, during site-specific alkyl modifications of oligonucleotides, it was feasible to fine-tune the overall hydrophobicity of the DNA, leading to development of cell-penetrating nucleic acids by ambiguously modulating the interactions between the DNAs and the lipid membranes. The cell-penetrating DNAs showed the potential for direct cytosolic drug delivery which can bypass an endocytic pathway with low efficiency, as demonstrated by delivery of siRNA and subsequent green fluorescent protein (GFP) knockdown in living cells. Secondly, inspired by the stimulus-triggered regulation of biological gene transcription, I developed ‘DNA-based molecular switches’ capable of controlling an activity of DNA polymerase upon chemical triggering. As an example, a Pb2+ activation technique, called lead-start isothermal amplification was demonstrated, which allows on-demand activation or deactivation of DNA polymerase at room temperature. The molecular switches were generated by systematically correlating the DNA polymerase inhibition by the TQ30 aptamer with Pb2+-responsive strand cleavage by the GR5 DNAzyme, and by employing various interconnecting strands, Pb2+ could effectively initiate or terminate the isothermal DNA amplification. Notably, the lead-start isothermal amplification exhibited remarkable specificity for Pb2+, significantly enhancing the DNA polymerase activity by 25-fold upon Pb2+ introduction. The switches could enable the quantitative analysis of isothermally amplified target genes in a parallel manner, as multiple reactions could be initiated and terminated at the same time under identical reaction conditions. Using a portable UV lamp and a smartphone camera, quantification of clinically relevant targets such as HPV type 16 genes in human serum was achieved with a detection limit as low as 0.1 nM, showing its suitability for point-of-care (PoC) testing in the field.