Molecular protectors: Superheroes for nanostructures

Abstract

Protecting complex nanostructures from degradation in physiological fluids while preserving their function remains a major limitation toward their implementation in biosensing and drug delivery. Using peptoid oligomers, Gang and coworkers protect DNA origami structures from Mg2+ depletion and enzymatic breakdown. Protecting complex nanostructures from degradation in physiological fluids while preserving their function remains a major limitation toward their implementation in biosensing and drug delivery. Using peptoid oligomers, Gang and coworkers protect DNA origami structures from Mg2+ depletion and enzymatic breakdown. The advent of nanotechnology has led to the ability to program the self-assembly of nanomaterials into well-defined structures that can be tailored for use in biomedical applications such as biosensing, drug delivery, and imaging.1Ramos A.P. Cruz M.A.E. Tovani C.B. Ciancaglini P. Biomedical applications of nanotechnology.Biophys. Rev. 2017; 9: 79-89Crossref PubMed Scopus (181) Google Scholar The rational design of nanostructures for desired applications requires precise control over their size, shape, and morphology. As seen in nature, the precise folding of biological molecules (i.e., peptide chains and nucleic acids) into their secondary structures is fundamental to their activity as the loss of this well-defined structure translates into a direct loss of functionality.2Zhu R.R. Wang W.R. Sun X.Y. Liu H. Wang S.L. Enzyme activity inhibition and secondary structure disruption of nano-TiO2 on pepsin.Toxicol. In Vitro. 2010; 24: 1639-1647Crossref PubMed Scopus (51) Google Scholar The vulnerability of nanostructures to the varying pH, enzyme activity, and ion concentrations inherent to physiological fluids limits their applications in biological systems.3Stephanopoulos N. Strategies for Stabilizing DNA Nanostructures to Biological Conditions.ChemBioChem. 2019; 20: 2191-2197Crossref PubMed Scopus (16) Google Scholar Therefore, the development of a versatile strategy to enhance the structural integrity of these nanomaterials without compromising their function is critical for physiological applications. We envision the use of adaptable “molecular protectors” to maintain the stability and function of nanostructures within in vivo systems in response to threats from various structural “destructors” (Figure 1). Current techniques used to stabilize nanostructures in physiological conditions include encapsulation, chemical cross-linking, protective coatings, backbone modifications, and variations in the types of culture media used.4Bila H. Kurisinkal E.E. Bastings M.M.C. Engineering a stable future for DNA-origami as a biomaterial.Biomater. Sci. 2019; 7: 532-541Crossref PubMed Google Scholar Heteropolymer-based coatings have been broadly applied to the protection of DNA. In one approach, Xu and coworkers designed a series of biomimetic random heteropolymers, composed of methyl methacrylate, oligo(ethylene glycol) methacrylate, 2-ethylhexyl methacrylate, and 3-sulfopropyl methacrylate, capable of co-assembly with target proteins, enhancing their stability and facilitating proper folding.5Panganiban B. Qiao B. Jiang T. DelRe C. Obadia M.M. Nguyen T.D. Smith A.A.A. Hall A. Sit I. Crosby M.G. et al.Random heteropolymers preserve protein function in foreign environments.Science. 2018; 359: 1239-1243Crossref PubMed Scopus (122) Google Scholar Shih and coworkers used an oligolysine and poly(ethylene glycol) copolymer to increase resistance to nuclease degradation while maintaining the overall topology of the nanostructure.6Ponnuswamy N. Bastings M.M.C. Nathwani B. Ryu J.H. Chou L.Y.T. Vinther M. Li W.A. Anastassacos F.M. Mooney D.J. Shih W.M. Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation.Nat. Commun. 2017; 8: 15654Crossref PubMed Scopus (233) Google Scholar The enhanced stability of DNA origami in physiological conditions has also been achieved by Linko and coworkers who utilized bovine serum albumin and class II hydrophobin protein coats to shield DNA origami from endonucleases, thereby enhancing its stability in vivo.7Auvinen H. Zhang H. Nonappa Kopilow A. Niemelä E.H. Nummelin S. Correia A. Santos H.A. Linko V. Kostiainen M.A. Protein Coating of DNA Nanostructures for Enhanced Stability and Immunocompatibility.Adv. Healthc. Mater. 2017; 6: 1-6Google Scholar Alternatively, Barisic and coworkers designed a polycationic-induced protective coat using chitosan and polyethyleneimine to protect DNA origami structures from both low salt concentrations and nucleolytic degradation.8Ahmadi Y. De Llano E. Barišić I. (Poly)cation-induced protection of conventional and wireframe DNA origami nanostructures.Nanoscale. 2018; 10: 7494-7504Crossref PubMed Google Scholar These studies show that preserving structural integrity is essential for the function of nanostructures in biological systems. As highlighted in this Matter preview, Gang and coworkers9Wang S.T. Gray M.A. Xuan S. Lin Y. Byrnes J. Nguyen A.I. Todorova N. Stevens M.M. Bertozzi C.R. Zuckermann R.N. Gang O. DNA origami protection and molecular interfacing through engineered sequence-defined peptoids.Proc. Natl. Acad. Sci. USA. 2020; 117: 6339-6348Crossref PubMed Scopus (57) Google Scholar used N-substituted glycines, a.k.a. peptoids, as a multifunctional coating for protecting DNA origami nanostructures. A common DNA origami motif, the wire-frame octahedron, was selected as a candidate for protection given its mechanical rigidity and use as a method to deliver molecular cargo trapped in its octahedral cage. DNA origami nanostructures are vulnerable to enzymatic attack, pH variation, and fluctuations in divalent cation (e.g., Mg2+) concentration, required to neutralize the negatively charged phosphate backbone of DNA. Relative to peptides, peptoids are highly resistant to enzymatic degradation while maintaining an intrinsic biocompatibility, making peptoid-based materials suitable for in vivo implementation. Gang and coworkers implemented molecular design principles to select a set of peptoid monomers as protective coatings. An N-(2-aminoethyl)glycine (Nae) residue was selected to introduce a cationic charge that electrostatically satisfies the negatively charged phosphodiester bond. In order to reduce the sensitivity to changes in pH and vulnerability to enzymatic degradation, Gang and coworkers used the neutral N-2-(2-(2 methoxyethoxy)ethoxy)ethylglycine (Nte) residue as a passivating layer. These monomers were arranged in both alternating (i.e., brush) and block order (Figures 2A and 2B ). Solid-phase peptoid synthesis offers monomer-level control over sequence order, which allowed Gang and coworkers to design molecular protectors with monomer-level precision to preserve DNA origami nanostructure function against common destructors such as Mg2+ depletion and enzymatic attack. The peptoid coatings PE1 and PE2 were highly effective in preserving DNA origami nanostructure function against structural destructors. Observation through transmission electron microscopy (TEM) suggested that peptoid-protected DNA origami nanostructures were shielded in low-Mg2+ (1.25 mM) solutions, demonstrating clearly defined characteristic edges and topology. Agarose gel electrophoresis of unprotected and protected origami indicated an electrophoretic band shift characteristic of damaged structures, which were significantly reduced in samples with higher concentrations of PE2. Dynamic light scattering experiments indicated broadening of size distributions upon the addition of ethylenediaminetetraacetic acid (EDTA) to unprotected origami, consistent with disintegration of the wire-frame structure due to EDTA’s strong chelating effect on metal ions. PE2-protected DNA origami nanostructures showed little broadening after EDTA addition, suggesting heightened stability against loss of Mg2+ to EDTA (Figure 2C). TEM studies on unprotected and protected DNA origami nanostructures incubated in solution with DNase I confirmed the effectiveness of the peptoid coating in preserving the wire-frame against enzymatic cleavage. Gang and coworkers suggested that steric shielding by Nte residues is effective in reducing DNase I binding. Notably, the peptoid coatings were effective in protecting molecular cargo encapsulated within the DNA origami nanostructures (Figure 2D). Effective shielding of BSA from hydrolytic attack via trypsin was confirmed using fluorescein-tagged bovine serum albumin (BSA) and encapsulation with both protected and unprotected DNA origami nanostructures. Through careful selection of peptoid monomers and sequence, molecular protectors for the stabilization of DNA origami nanostructures against the destructors of Mg2+ depletion and DNase attack and the safeguarding of molecular cargo were designed. With recent progress in the rational design and controlled synthesis of nanomaterials with tunable properties, there is an increased focus on translating their use as drug delivery vehicles, biosensors, and imaging agents. To ensure the stability of nanostructures in the face of damage by destructors in physiological conditions, molecular protectors have been developed to maintain structural integrity, which is intimately linked to function. Future nanomaterials need to be codeveloped with complimentary protection strategies to facilitate applications in complex biological systems. Given the diversity of nanostructures and potential destructors, a universal “superhero-like material” to effectively stabilize all biologically functional nanostructures is impractical. Instead, we foresee opportunities in the development of combinatorial molecular protector libraries and machine learning techniques to identify appropriate methods of protection based on the desired shape, topology, and application of a particular nanomaterial.10Sanchez-Lengeling B. Aspuru-Guzik A. Inverse molecular design using machine learning: Generative models for matter engineering.Science. 2018; 361: 360-365Crossref PubMed Scopus (634) Google Scholar The ongoing investigations into methods of increasing the biostability of nanostructures will allow for the realization of the full potential of nanotechnology in biomedical applications. We acknowledge the support of the Natural Sciences and Engineering Research Council of Canada (NSERC), RGPIN-2021-03554 . A.U. thanks the University of Toronto for the Faculty of Arts & Science Top (FAST) Doctoral Fellowship. We also gratefully acknowledge the Department of Chemistry and the Department of Chemical Engineering & Applied Chemistry at the University of Toronto for their support.