Exceptional biostability of paranemic crossover (PX) DNA, crossover-dependent nuclease resistance, and implications for DNA nanotechnology

AbstractInherent nanometer-sized features and molecular recognition properties make DNA a useful material in constructing nanoscale objects, with alluring applications in biosensing and drug delivery. However, DNA can be easily degraded by nucleases present in biological fluids, posing a considerable roadblock to realizing the full potential of DNA nanotechnology for biomedical applications. Here we investigated the nuclease resistance and biostability of the multi-stranded motif called paranemic crossover (PX) DNA and discovered a remarkable and previously unreported resistance to nucleases. We show that PX DNA has more than an order of magnitude increased resistance to degradation by DNase I, serum, and urine compared to double stranded DNA. We further demonstrate that the degradation resistance decreases monotonically as DNA crossovers are removed from the structure, suggesting that frequent DNA crossovers disrupt either the binding or catalysis of nucleases or both. Further, we show using mouse and human cell lines that PX DNA does not affect cell proliferation or interfere with biological processes such as myogenesis. These results have important implications for building DNA nanostructures with enhanced biostability, either by adopting PX-based architectures or by carefully engineering crossovers. We contend that such crossover-dependent nuclease resistance could potentially be used to add “tunable biostability” to the many features of DNA nanotechnology.

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Cellular processing and destinies of artificial DNA nanostructures

Chemical Society Reviews ◽

10.1039/c5cs00700c ◽

2016 ◽

Vol 45 (15) ◽

pp. 4199-4225 ◽

Cited By ~ 87

Author(s):

Di Sheng Lee ◽

Hang Qian ◽

Chor Yong Tay ◽

David Tai Leong

Keyword(s):

Dna Nanotechnology ◽

Dna Nanostructures ◽

Panoramic View ◽

Artificial Dna ◽

Cellular Processing ◽

Mechanistic Understanding ◽

The Many ◽

In Cells

This review gives a panoramic view of the many DNA nanotechnology applications in cells, mechanistic understanding of how and where their interactions occur and their subsequent outcomes.

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Engineering DNA nanotubes for resilience in an E. coli TXTL system

Synthetic Biology ◽

10.1093/synbio/ysy001 ◽

2018 ◽

Vol 3 (1) ◽

Cited By ~ 5

Author(s):

Melissa A Klocke ◽

Jonathan Garamella ◽

Hari K K Subramanian ◽

Vincent Noireaux ◽

Elisa Franco

Keyword(s):

Deoxyribonucleic Acid ◽

Dna Nanotechnology ◽

Dna Degradation ◽

Dna Nanostructures ◽

E Coli ◽

Physiological Conditions ◽

Double Stranded Dna ◽

Dna Nanotubes ◽

Sticky End ◽

Dna Nanotube

Abstract Deoxyribonucleic acid (DNA) nanotechnology is a growing field with potential intracellular applications. In this work, we use an Escherichia coli cell-free transcription–translation (TXTL) system to assay the robustness of DNA nanotubes in a cytoplasmic environment. TXTL recapitulates physiological conditions as well as strong linear DNA degradation through the RecBCD complex, the major exonuclease in E. coli. We demonstrate that chemical modifications of the tiles making up DNA nanotubes extend their viability in TXTL for more than 24 h, with phosphorothioation of the sticky end backbone being the most effective. Furthermore, we show that a Chi-site double-stranded DNA, an inhibitor of the RecBCD complex, extends DNA nanotube lifetime significantly. These complementary approaches are a first step toward a systematic prototyping of DNA nanostructures in active cell-free cytoplasmic environments and expand the scope of TXTL utilization for bioengineering.

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DNA origami protection and molecular interfacing through engineered sequence-defined peptoids

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1919749117 ◽

2020 ◽

Vol 117 (12) ◽

pp. 6339-6348 ◽

Cited By ~ 19

Author(s):

Shih-Ting Wang ◽

Melissa A. Gray ◽

Sunting Xuan ◽

Yiyang Lin ◽

James Byrnes ◽

...

Keyword(s):

Biomedical Applications ◽

Electrostatic Interactions ◽

Biological Fluids ◽

Structural Characteristics ◽

Dna Nanotechnology ◽

Dna Origami ◽

Cell Targeting ◽

The Stability ◽

Molecular Plasticity

DNA nanotechnology has established approaches for designing programmable and precisely controlled nanoscale architectures through specific Watson−Crick base-pairing, molecular plasticity, and intermolecular connectivity. In particular, superior control over DNA origami structures could be beneficial for biomedical applications, including biosensing, in vivo imaging, and drug and gene delivery. However, protecting DNA origami structures in complex biological fluids while preserving their structural characteristics remains a major challenge for enabling these applications. Here, we developed a class of structurally well-defined peptoids to protect DNA origamis in ionic and bioactive conditions and systematically explored the effects of peptoid architecture and sequence dependency on DNA origami stability. The applicability of this approach for drug delivery, bioimaging, and cell targeting was also demonstrated. A series of peptoids (PE1–9) with two types of architectures, termed as “brush” and “block,” were built from positively charged monomers and neutral oligo-ethyleneoxy monomers, where certain designs were found to greatly enhance the stability of DNA origami. Through experimental and molecular dynamics studies, we demonstrated the role of sequence-dependent electrostatic interactions of peptoids with the DNA backbone. We showed that octahedral DNA origamis coated with peptoid (PE2) can be used as carriers for anticancer drug and protein, where the peptoid modulated the rate of drug release and prolonged protein stability against proteolytic hydrolysis. Finally, we synthesized two alkyne-modified peptoids (PE8 and PE9), conjugated with fluorophore and antibody, to make stable DNA origamis with imaging and cell-targeting capabilities. Our results demonstrate an approach toward functional and physiologically stable DNA origami for biomedical applications.

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Structural DNA Nanotechnology: Artificial Nanostructures for Biomedical Research

Annual Review of Biomedical Engineering ◽

10.1146/annurev-bioeng-062117-120904 ◽

2018 ◽

Vol 20 (1) ◽

pp. 375-401 ◽

Cited By ~ 41

Author(s):

Yonggang Ke ◽

Carlos Castro ◽

Jong Hyun Choi

Keyword(s):

Biomedical Research ◽

Self Assembly ◽

Biomedical Applications ◽

Dna Nanotechnology ◽

Dna Nanostructures ◽

Considerable Effort ◽

Artificial Nanostructures ◽

Active Pursuit ◽

External Stimuli ◽

Structural Dna Nanotechnology

Structural DNA nanotechnology utilizes synthetic or biologic DNA as designer molecules for the self-assembly of artificial nanostructures. The field is founded upon the specific interactions between DNA molecules, known as Watson–Crick base pairing. After decades of active pursuit, DNA has demonstrated unprecedented versatility in constructing artificial nanostructures with significant complexity and programmability. The nanostructures could be either static, with well-controlled physicochemical properties, or dynamic, with the ability to reconfigure upon external stimuli. Researchers have devoted considerable effort to exploring the usability of DNA nanostructures in biomedical research. We review the basic design methods for fabricating both static and dynamic DNA nanostructures, along with their biomedical applications in fields such as biosensing, bioimaging, and drug delivery.

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Strategies to Build Hybrid Protein–DNA Nanostructures

Nanomaterials ◽

10.3390/nano11051332 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1332

Author(s):

Armando Hernandez-Garcia

Keyword(s):

Dynamic Systems ◽

Dna Nanotechnology ◽

Chemical Properties ◽

Hybrid Nanostructures ◽

Advanced Materials ◽

Dna Nanostructures ◽

Hybrid Protein ◽

Structural Protein ◽

The Individual ◽

Structural Dna Nanotechnology

Proteins and DNA exhibit key physical chemical properties that make them advantageous for building nanostructures with outstanding features. Both DNA and protein nanotechnology have growth notably and proved to be fertile disciplines. The combination of both types of nanotechnologies is helpful to overcome the individual weaknesses and limitations of each one, paving the way for the continuing diversification of structural nanotechnologies. Recent studies have implemented a synergistic combination of both biomolecules to assemble unique and sophisticate protein–DNA nanostructures. These hybrid nanostructures are highly programmable and display remarkable features that create new opportunities to build on the nanoscale. This review focuses on the strategies deployed to create hybrid protein–DNA nanostructures. Here, we discuss strategies such as polymerization, spatial directing and organizing, coating, and rigidizing or folding DNA into particular shapes or moving parts. The enrichment of structural DNA nanotechnology by incorporating protein nanotechnology has been clearly demonstrated and still shows a large potential to create useful and advanced materials with cell-like properties or dynamic systems. It can be expected that structural protein–DNA nanotechnology will open new avenues in the fabrication of nanoassemblies with unique functional applications and enrich the toolbox of bionanotechnology.

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Biomedical Uses of Sulfobetaine-Based Zwitterionic Materials

Organic Materials ◽

10.1055/s-0040-1721741 ◽

2020 ◽

Vol 02 (04) ◽

pp. 342-357

Author(s):

Francesco Zaccarian ◽

Matthew B. Baker ◽

Matthew J. Webber

Keyword(s):

Biomedical Applications ◽

Foreign Body Reaction ◽

Inflammatory Responses ◽

Cellular Adhesion ◽

Review Of The Literature ◽

Future Prospects ◽

Protein Fouling ◽

Biomedical Device ◽

The Many ◽

Hydrophilic Coatings

Protein fouling can render a biomedical device dysfunctional, and also serves to nucleate the foreign body reaction to an implanted material. Hydrophilic coatings have emerged as a commonly applied route to combat interface-mediated complications and promote device longevity and limited inflammatory response. While polyethylene glycol has received a majority of the attention in this regard, coatings based on zwitterionic moieties have been more recently explored. Sulfobetaines in particular constitute one such class of zwitterions explored for use in mitigating surface fouling, and have been shown to reduce protein adsorption, limit cellular adhesion, and promote increased functional lifetimes and limited inflammatory responses when applied to implanted materials and devices. Here, we present a focused review of the literature surrounding sulfobetaine, beginning with an understanding of its chemistry and the methods by which it is applied to the surface of a biomedical device in molecular and polymeric forms, and then advancing to the many early demonstrations of function in a variety of biomedical applications. Finally, we provide some insights into the benefits and challenges presented by its use, as well as some outlook on the future prospects for using this material to improve biomedical device practice by addressing interface-mediated complications.

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Hybrid Nanoassemblies from Viruses and DNA Nanostructures

Nanomaterials ◽

10.3390/nano11061413 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1413

Author(s):

Sofia Ojasalo ◽

Petteri Piskunen ◽

Boxuan Shen ◽

Mauri A. Kostiainen ◽

Veikko Linko

Keyword(s):

Cell Activation ◽

Immune Cell ◽

Dna Nanotechnology ◽

Biological Properties ◽

Dna Nanostructures ◽

Dna Structures ◽

Nanoscale Structures ◽

Immune Cell Activation ◽

Wide Range ◽

Structural Dna Nanotechnology

Viruses are among the most intriguing nanostructures found in nature. Their atomically precise shapes and unique biological properties, especially in protecting and transferring genetic information, have enabled a plethora of biomedical applications. On the other hand, structural DNA nanotechnology has recently emerged as a highly useful tool to create programmable nanoscale structures. They can be extended to user defined devices to exhibit a wide range of static, as well as dynamic functions. In this review, we feature the recent development of virus-DNA hybrid materials. Such structures exhibit the best features of both worlds by combining the biological properties of viruses with the highly controlled assembly properties of DNA. We present how the DNA shapes can act as “structured” genomic material and direct the formation of virus capsid proteins or be encapsulated inside symmetrical capsids. Tobacco mosaic virus-DNA hybrids are discussed as the examples of dynamic systems and directed formation of conjugates. Finally, we highlight virus-mimicking approaches based on lipid- and protein-coated DNA structures that may elicit enhanced stability, immunocompatibility and delivery properties. This development also paves the way for DNA-based vaccines as the programmable nano-objects can be used for controlling immune cell activation.

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Constructing Large 2D Lattices Out of DNA-Tiles

Molecules ◽

10.3390/molecules26061502 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1502

Author(s):

Johannes M. Parikka ◽

Karolina Sokołowska ◽

Nemanja Markešević ◽

J. Jussi Toppari

Keyword(s):

Self Assembly ◽

Dna Nanotechnology ◽

Building Blocks ◽

High Yield ◽

Dna Nanostructures ◽

Liquid Interfaces ◽

Basic Principles ◽

Dna Tiles ◽

One Step ◽

Solid Liquid

The predictable nature of deoxyribonucleic acid (DNA) interactions enables assembly of DNA into almost any arbitrary shape with programmable features of nanometer precision. The recent progress of DNA nanotechnology has allowed production of an even wider gamut of possible shapes with high-yield and error-free assembly processes. Most of these structures are, however, limited in size to a nanometer scale. To overcome this limitation, a plethora of studies has been carried out to form larger structures using DNA assemblies as building blocks or tiles. Therefore, DNA tiles have become one of the most widely used building blocks for engineering large, intricate structures with nanometer precision. To create even larger assemblies with highly organized patterns, scientists have developed a variety of structural design principles and assembly methods. This review first summarizes currently available DNA tile toolboxes and the basic principles of lattice formation and hierarchical self-assembly using DNA tiles. Special emphasis is given to the forces involved in the assembly process in liquid-liquid and at solid-liquid interfaces, and how to master them to reach the optimum balance between the involved interactions for successful self-assembly. In addition, we focus on the recent approaches that have shown great potential for the controlled immobilization and positioning of DNA nanostructures on different surfaces. The ability to position DNA objects in a controllable manner on technologically relevant surfaces is one step forward towards the integration of DNA-based materials into nanoelectronic and sensor devices.

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Spatiotemporally programmable cascade hybridization of hairpin DNA in polymeric nanoframework for precise siRNA delivery

Nature Communications ◽

10.1038/s41467-021-21442-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Feng Li ◽

Wenting Yu ◽

Jiaojiao Zhang ◽

Yuhang Dong ◽

Xiaohui Ding ◽

...

Keyword(s):

Steric Effect ◽

Sirna Delivery ◽

Dna Nanotechnology ◽

Hybridization Chain Reaction ◽

Dna Nanostructures ◽

Chain Reaction ◽

Dna Hairpins ◽

In Vivo Experiments ◽

Dna And Rna

AbstractDNA nanostructures have been demonstrated as promising carriers for gene delivery. In the carrier design, spatiotemporally programmable assembly of DNA under nanoconfinement is important but has proven highly challenging due to the complexity–scalability–error of DNA. Herein, a DNA nanotechnology-based strategy via the cascade hybridization chain reaction (HCR) of DNA hairpins in polymeric nanoframework has been developed to achieve spatiotemporally programmable assembly of DNA under nanoconfinement for precise siRNA delivery. The nanoframework is prepared via precipitation polymerization with Acrydite-DNA as cross-linker. The potential energy stored in the loops of DNA hairpins can overcome the steric effect in the nanoframework, which can help initiate cascade HCR of DNA hairpins and achieve efficient siRNA loading. The designer tethering sequence between DNA and RNA guarantees a triphosadenine triggered siRNA release specifically in cellular cytoplasm. Nanoframework provides stability and ease of functionalization, which helps address the complexity–scalability–error of DNA. It is exemplified that the phenylboronate installation on nanoframework enhanced cellular uptake and smoothed the lysosomal escape. Cellular results show that the siRNA loaded nanoframework down-regulated the levels of relevant mRNA and protein. In vivo experiments show significant therapeutic efficacy of using siPLK1 loaded nanoframework to suppress tumor growth.

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Oligonucleotides carrying nucleoside antimetabolites as potential prodrugs

Current Medicinal Chemistry ◽

10.2174/0929867328666211129124039 ◽

2021 ◽

Vol 28 ◽

Author(s):

Carme Fàbrega ◽

Anna Clua ◽

Ramon Eritja ◽

Anna Aviñó

Keyword(s):

Antisense Oligonucleotides ◽

Biomedical Applications ◽

Enzymatic Degradation ◽

Research Effort ◽

Synergetic Effect ◽

Chemotherapeutic Agents ◽

Dna Nanostructures ◽

Chemically Modified ◽

Near Future ◽

Large Research

Background: Nucleoside and nucleobase antimetabolites are an important class of chemotherapeutic agents for the treatment of cancer as well as other diseases. Introduction: In order to avoid undesirable side effects, several prodrug strategies have been developed for that purpose. In the present review, we describe a relatively unknown strategy that consists in the use of oligonucleotides modified with nucleoside antimetabolites as prodrugs. Method: The active nucleotides are generated by enzymatic degradation once incorporated into cells. This strategy has attracted large interest and is very active at present due to the continuous developments made on therapeutic oligonucleotides and the recent advances in the field of nanomaterials and nanomedicine. Results: A large research effort was done mainly in the improvement of the antiproliferative properties of nucleoside homopolymers, but recently, chemically modified aptamers, antisense oligonucleotides and/or siRNA carrying antiproliferative nucleotides have demonstrated a great potential due to the synergetic effect of both therapeutic entities. In addition, DNA nanostructures with interesting properties have been built to combine antimetabolites and enhancers of cellular uptake in the same scaffold. Finally, protein nanoparticles functionalized with receptor-binders and antiproliferative oligomers represent a new avenue for a more effective treatment in cancer therapy. Conclusion: It is expected that oligonucleotides carrying nucleoside antimetabolites will be considered as potential drugs in the near future for biomedical applications.

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