Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation

Abstract

DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications.

Protein Coating of DNA Nanostructures for Enhanced Stability and Immunocompatibility

“…[44] Moreover, it has been suggested that the delivery rates could be improved by employing DNA intercalators, [45] by directly incorporating specific proteins into the structures, [46] or by using cationic polymer coating. [47][48][49] For DNA binding and coating purposes, dendrons and dendrimers are ideal polymer structures. [50,51] They are regularly branched synthetic molecules with essentially monodisperse structure [52] and high density of functional groups capable of binding various biomolecules with high affinity through multivalent and multimodal interactions.…”

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

“…[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. [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 nanostructures investigated in this work, that is,t he Rothemund triangle, [1] a2 4-helix bundle (24HB), [15] and asix-helix bundle (6HB). [12][13][14] All the more surprising was the discovery that DNAorigami are stable in water for several weeks.…”

“…[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. [12][13][14] All the more surprising was the discovery that DNAorigami are stable in water for several weeks. [12][13][14] All the more surprising was the discovery that DNAorigami are stable in water for several weeks.…”

“…[12][13][14] All the more surprising was the discovery that DNAorigami are stable in water for several weeks. [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. However,other studies observed DNAorigami denaturation in buffers containing low-Mg 2+ concentrations.…”

“…[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.…”