On the Stability of DNA Origami Nanostructures in Low‐Magnesium Buffers

Abstract

DNAo rigami structures have great potential as functional platforms in various biomedical applications.Many applications,h owever,a re incompatible with the high Mg 2+ concentrations commonly believed to be ap rerequisite for maintaining DNAo rigami integrity.H erein, we investigate DNAo rigami stability in low-Mg 2+ buffers.D NA origami stability is found to crucially depend on the availability of residual Mg 2+ ions for screening electrostatic repulsion. The presence of EDTAa nd phosphate ions may thus facilitate DNAo rigami denaturation by displacing Mg 2+ ions from the DNAb ackbone and reducing the strength of the Mg 2+ -DNA interaction, respectively.M ost remarkably,t hese buffer dependencies are affected by DNAo rigami superstructure. However,b yr ationally selecting buffer components and considering superstructure-dependent effects,t he structural integrity of ag iven DNAo rigami nanostructure can be maintained in conventional buffers even at Mg 2+ concentrations in the low-micromolar range.DNA origami [1,2] has become awidely used method for the fabrication of complex, yet well-defined nanostructures [3] with applications in biophysics, [4] molecular biology, [5] and drug and enzyme delivery. [6] Since many of these applications rely on intact DNAn anostructures,t he investigation of DNA origami stability under application-specific conditions has become am ajor research focus. [7][8][9][10][11] Biomedical applications in particular are often incompatible with the comparatively high (10-20 mm)M g 2+ concentrations required for DNA origami assembly.Onthe other hand, low-Mg 2+ concentration ( 1mm)h as been identified as one of the two most critical parameters that reduce DNAorigami stability in cell culture media. [8] Consequently,many approaches have been reported for protecting DNAo rigami nanostructures against destabilizing conditions and in particular low-Mg 2+ concentrations. [12][13][14] All the more surprising was the discovery that DNAorigami are stable in water for several weeks. [10] In these experiments,t he Mg 2+ -containing assembly buffer was exchanged for water through spin filtering,r esulting in Mg 2+ concentrations of afew micromolar.Although not completely Mg 2+ -free,m any applications may benefit from intact DNA origami nanostructures under such low-Mg 2+ conditions. However,other studies observed DNAorigami denaturation in buffers containing low-Mg 2+ concentrations. [7,8,11,12,14] These discrepancies could have various origins,s uch as differences in the buffer-exchange methods,buffer conditions, and DNAo rigami designs.I nt his work, we therefore investigate the stability of three DNAorigami nanostructures in as election of low-Mg 2+ buffers using the spin filteringbased buffer-exchange approach established by Linko et al. [10] We find that the composition of the buffer plays acritical role in DNAo rigami stability,w hile different DNAo rigami nanostructures show different buffer dependencies.First, we set out to reproduce the results of Linko et al. for the three DNAo rigami nanost...

Real‐Time Observation of Superstructure‐Dependent DNA Origami Digestion by DNase I Using High‐Speed Atomic Force Microscopy

“…A particularly important issue for medicala pplications is their interaction with proteins such as endonucleases, which may degrade the well-defined nanoscale shapes.H erein, fundamental insights into this interaction are provided by monitoring DNase Id igestion of four structurally distinct DNA origami nanostructures (DONs) in real time and at as ingle-structure level by using high-speed atomic force microscopy.T he effect of the solid-liquidi nterface on DON digestioni sa lso assessed by comparison with experiments in bulk solution. [16, 17] Although it has been shown that DONs can survive under low-cation conditions, [18,19] stability studies performedi n serum or cell culture media have yielded somewhat controversial and quite distinct results. [2][3][4][5] In recenty ears, programmable DNA origami nanostructures (DONs) [6][7][8][9][10] have been considered promising candidates for the development of tailored and multifunctional drug-delivery vehicles.…”

“…[11][12][13][14] For therapeutic in vivo applications, the DON vehicles should preferably be compatible with the immunes ystem, resistant to nucleases, have as ufficiently long circulation half-life, and be able to maintain their shape at the ionic strengths of the biological fluids. [16, 17] Although it has been shown that DONs can survive under low-cation conditions, [18,19] stability studies performedi n serum or cell culture media have yielded somewhat controversial and quite distinct results. [16, 17] Although it has been shown that DONs can survive under low-cation conditions, [18,19] stability studies performedi n serum or cell culture media have yielded somewhat controversial and quite distinct results.…”

“…[15] However,c oncerns have been raised regarding the stabilityo fD ONs and their performance in biological media. [8,15,[19][20][21] Therefore, significant efforts have been madei nto coating ands tabilizing DONs under biologically relevant conditions. [8,15,[19][20][21] Therefore, significant efforts have been madei nto coating ands tabilizing DONs under biologically relevant conditions.…”

“…[15] However,c oncerns have been raised regarding the stabilityo fD ONs and their performance in biological media. [16,17] Although it has been shown that DONs can survive under low-cation conditions, [18,19] stability studies performedi n serum or cell culture media have yielded somewhat controversial and quite distinct results. [8,15,[19][20][21] Therefore, significant efforts have been madei nto coating ands tabilizing DONs under biologically relevant conditions.…”

“…[16,17] Although it has been shown that DONs can survive under low-cation conditions, [18,19] stability studies performedi n serum or cell culture media have yielded somewhat controversial and quite distinct results. [8,15,[19][20][21] Therefore, significant efforts have been madei nto coating ands tabilizing DONs under biologically relevant conditions. [17,[22][23][24][25] Herein, we study DON digestion by endonucleases (DNase I) in dependence of DON superstructure.…”