DNA nanopores offer a unique nano-scale foothold at the membrane interface that can help advance the life sciences as biophysical research tools or gate-keepers for drug delivery. Biological applications require sufficient physiological stability and membrane activity for viable biological action. In this report, we determine essential parameters for efficient nanopore folding and membrane binding in biocompatible cell media. The parameters are identified for an archetypal DNA nanopore composed of six interwoven strands carrying cholesterol lipid anchors. Using gel electrophoresis and fluorescence spectroscopy, the nanostructures are found to assemble efficiently in cell media, such as LB and DMEM, and remain structurally stable at physiological temperatures. Furthermore, the pores’ oligomerization state is monitored using fluorescence spectroscopy and confocal microscopy. The pores remain predominately water-soluble over 24 h in all buffer systems, and were able to bind to lipid vesicles after 24 h to confirm membrane activity. However, the addition of fetal bovine serum to DMEM causes a significant reduction in nanopore activity. Serum proteins complex rapidly to the pore, most likely via ionic interactions, to reduce the effective nanopore concentration in solution. Our findings outline crucial conditions for maintaining lipidated DNA nanodevices, structurally and functionally intact in cell media, and pave the way for biological studies in the future.
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