Structural and Functional Stability of DNA Nanopores in Biological Media

Abstract

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.

Design, assembly, and characterization of membrane-spanning DNA nanopores

“…In order for DNA nanostructures to insert into lipid bilayers, hydrophobic groups must be incorporated to act as lipid anchors 44 . However, introducing hydrophobic anchors increases the synthesis complexity and can lead to aggregation of pores 36,[45][46][47][48] . Other drawbacks are related to the structure's flexibility, and the permeability of the pore's walls to ions.…”

“…For directly visualizing DNA nanopores, transmission electron microscopy (TEM) (steps [17][18][19][20][21][22][23][24][25][26] and AFM (steps 27-40) can be used. The pores' membrane interaction and insertion can be probed with TEM and AFM, and a gel binding assay (steps [41][42][43][44][45][46][47][48][49][50][51]. Finally, DNA pore function can be confirmed by single-channel current recordings (steps 52-72) and dye flux assays (steps [73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90].…”

Controlling aggregation of cholesterol-modified DNA nanostructures

“…Previous approaches to prevent cholesterol-induced aggregation of DNA nanostructures were limited to reducing the number of cholesterol tags (26), switching to an alternative membrane anchoring strategy using streptavidin as a linker between biotinylated DNA and lipids (18), or employing surfactants (32). However, fewer cholesterol modifications diminish membrane interaction (19), a streptavidin–biotin linkage is not applicable for studying biological cell membranes and their mimics, and surfactants are likely to impair the structure and integrity of lipid bilayers.…”

Editorial for the Special Issue on “Nucleic Acid Architectures for Therapeutics, Diagnostics, Devices and Materials”

“…This special issue assembles 11 original articles (with seven research manuscripts and four reviews) which nicely outline several advances made in the field of RNA and DNA nanotechnology. Unified by the versatile use of the intriguing biopolymers, these manuscripts explore the various facets of nucleic acid nanostructures such as design, production, and characterization of RNA and DNA nanoassemblies [1,2,3,4], rational design of functional molecular machines [5,6,7,8], immunorecognition of nucleic acid nanoparticles [8], in vivo delivery of therapeutic nucleic acids [9,10], and nucleic acid-based biosensors [11]. We anticipate this special issue to be accessible to a wide audience, as it explores not only the biological aspects of nucleic acid nanodesigns, but also different methodologies of their production, their interactions with other classes of biological molecules, physicochemical characteristics, and possible applications.…”

“…One such device is the DNA nanopore, which offers a unique membrane-bound tool for furthering biophysical research and biosensing. In this issue, Burns and Howorka describe the design and evaluation of a DNA nanopore in various biological milieu [5]. The authors identify parameters for the assembly of a hexameric DNA nanopore using gel electrophoresis and fluorescence spectroscopy, and further confirm its integrity in cell media, as well as its successful integration into lipid membranes.…”