Bioengineering multifunctional DNA nanomaterials – a molecular playground

The influence of DNA nanotechnology for innovations in personalized medicine
Bioengineering multifunctional DNA nanomaterials – a molecular playground

Having recently written about epigenetic mechanisms underlying oncogenesis and about epigenetic engineering associated with disease evolution. In this post, I briefly explore the increasingly popular concept of bioengineering multifunctional DNA nanomaterials. The idea broadly encompasses the interdisciplinary fields of materials science, bioengineering, and medicine. Standout developments of the technique include DNA nanocarriers, nanoswitches, and multifunctional nanomaterials engineered at nanoscale precision by using pioneering techniques, to facilitate interventional strategies in personalized medicine (figure 1).

While DNA inherently stores genetic information in biological systems, DNA nanotechnology goes beyond the biological context of the molecule, to assemble and connect structural motifs via molecular self-assembly to facilitate a bench-to-clinic framework in nanomedicine [Seeman 2017]. DNA is highly flexible as a single strand and demonstrates increased rigidity in its double strand form and is therefore well-suited as a nanoscale building block [Bustamante 2003]. DNA nanoarchitectures are broadly classified to three main areas – DNA tetrahedra, DNA origami, and DNA devices. This post is broadly about the origin of DNA nanotechnology and its wide-ranging applications that provide ample incentive for innovations in the field. 

Figure 1: A schematic representation of the fate of DNA nanomaterials including tetrahedral DNA nanostructures (TDNs) and plasmid DNA (pDNA) following their delivery into cells. Nucleic acid nanomaterials can enter cells to be internalized and degraded via lysosomes. To prevent lysosomal degradation when inside the cells, the vector-DNA material complexes can be bioengineered as molecules embedded in an endosome to create nucleic acid cargo [Ma 2021].

The origins of DNA origami

The origins of using DNA as a biomaterial point back to 1982, when biologist Nadrian Seeman pioneered structural DNA nanotechnology. The concept maximized on Watson-Crick base-pairing to create covalent links with enormous specificity in order to engineer covalently joined 3-D networks of nucleic acids [Seeman 1982]. This led to tremendous interests in DNA structures thereon and enabled the formation of lattices modified with sticky ends to bioengineer DNA nanostructures that eventually led to DNA origami (figure 2). 

In 2004, biochemist William Shih played up on this idea and constructed an octahedron using single stranded DNA based on a two-step procedure to assemble a giant branched molecule with bulging arms in an octahedron-shaped nanostructure [Shih 2004]. In 2006, computational biologist Paul Rothemund first reported the concept of DNA origami technology to explore ‘bottom-up fabrication’ and directed the self-organization of atoms and molecules to build relatively simple nanostructures of high complexity [Rothemund 2006], for delivery into cells.

Several years later, Shih and teams built-up on DNA origami to form high-precision personalized cancer vaccines delivered to administer immunotherapies in the form of implantable or injectable biomaterial-based scaffolds. These materials can train a patient’s immune system via dendritic cell activation and cytotoxic T-cell response, to attack cancerous tumors, including melanomas with promising initial-stage clinical trial outcomes [Maurer 2020].

Figure 2: DNA self-assembly strategies. (A) DNA objects, (B) DNA motifs in assembled arrays, (C) DNA origami strategies, and (D) DNA brick strategies to create nanostructures [Chandrasekaran 2019].

Iconic DNA as a construction material

Precision medicine is based on disease-specific molecular classifications that accurately reflect clinical behavior [Collins 2015]. Increasingly, large-scale clinical analyses are being conducted on multidimensional molecules including DNA/RNA proteins and small molecules, to better classify and understand disease through genomics, transcriptomics, and proteomics [Kristensen 2014]. Meanwhile structural DNA nanotechnology can regulate the fate of stem cells [Cheung 2016], facilitate the delivery of nucleic acid-based drugs [Lv 2021], or develop DNA framework-based cancer diagnosis (figure 3) [Yin 2023] to prove its multifunctionality in medicine [Chandrasekaran 2019].

Biological applications of DNA nanostructures cover a few topics of personal research interest (and experience too), to notably include cardiac muscle tissue engineering, neural tissue engineering, bone tissue engineering, and skin tissue engineering applications. The strategy is also well-defined for drug delivery, cancer vaccines, and tumor therapy in biomedicine.

Figure 3: Developing immunotherapy vaccines by leveraging DNA origami nanotechnology and immune activators to stimulate stronger and long-lasting immune responses against cancer and potentially infectious diseases. Image credit: Wyss Institute

Furthermore, DNA intelligence nanodevices known as ‘nanocentipedes’ and nanobots function as multivalent drug carriers to activate a series of related cascade reactions in target cells to produce therapeutic effects in cancer medicine [Li 2016]. Added efforts include delivering biomedical diagnosis in the form of microRNAs that are increasingly interpreted as more efficient biomarkers of disease [Chandrasekaran 2019].

DNA-based designs in the lab: Tensegrity and Lab-on-a-Molecule

DNA nanotechnology designs in the lab include combining the flexibility of single-strand DNA with multilayered nanostructures to build tensegrity structures – so named for their tensional integrity [Liedl 2010]. These constructs are typically derived to confer material stability with characteristically high strength-to-weight ratios that offer great resilience to engineer nanoarchitectural platforms. Tensegrity has heavily influenced the first principles behind emulating an organ-like microenvironment on an organ-chip instrument in the field of mechanobiology, which increasingly seeks to replace animal models to advance personalized medicine [Ingber 2014, Bhatia 2014]. 

[Movie 1: Nanoswitch Technology: Testing Molecular Modulators from Wyss Institute on Vimeo.  Nanoswitch technology – testing molecular modulators. DNA nanoswitches can be used to characterize molecules that stabilize the binding of two disease-relevant targets, which causes the nanoswitch to loop and slow the movement in gel electrophoresis. Credit: Wyss Institute.]

These ventures have led to the development of bioinspired DNA nanoswitches to create lab-on-a-molecule devices to investigate molecular interactions [Koussa 2015] (movie 1). Nanoswitches present a simple yet innovative, instrument-free platform only with gel-electrophoresis-based analysis of DNA self-assembly, to determine molecular associations and dissociations. By functionalizing DNA oligonucleotides with molecules of interest hybridized to specific locations on a single-strand DNA scaffold, biologists can resolve the interaction states of the DNA, which appear as distinct bands on gel electrophoresis to measure multistate molecular kinetics (movie 1).

How to create a DNA nanostructure framework

The methods discussed thus far have shown great promise in tumor targeted therapy and several pathological courses. The form of tetrahedral DNA nanostructures [Ma 2021] are vital for drug discovery, regenerative and multifunctional applications since it can resist deformation under enzymatic digestion [Shao 2017]. Bioengineers have also coated DNA nanostructures with PEG-oligolysines and chemically cross-linked them to increase their resistance to enzymatic degradation for enhanced stability in vivo (figure 4). The generic method of customized DNA origami or DNA nanostructure preparation includes a seven-step framework:

  • Composing the nanostructure of an object
  • Creating a scaffold style layout to define the DNA sequence by using software
  • Designing oligoes of the scaffold and staple
  • Facilitating DNA self-assembly in a buffer solution
  • Purifying and functionalizing the DNA nanostructure
  • Analyzing and visualizing the construct
  • Expanding and exploring its applications

DNA nanotechnology confers high biocompatibility, structural diversity, and low cytotoxicity, for efficient drug delivery and therapeutic functions. Some of these nanostructures are currently in use for tumor therapy, and their methods of administration and applications are listed on table 1, below [Ma 2021].

Figure 4: Preventing DNA degradation by coating DNA nanostructures with PEG-oligolysines. Then chemically crosslinking the distinct PEG-oligolysines with glutaraldehyde as a crosslinking agent to resist degradation when exposed to DNA nuclease enzymes for overall stability. Credit: Frances Anastassacos Wyss Institute at Harvard University.

Pioneering DNA nanotechnologies in tissue engineering, biosensing and biomedical diagnosis. 

To wrap things up with a more personal viewpoint, how exactly does DNA nanotechnology influence tissue engineering? During conventional tissue engineering practices, tissue regeneration depends on cell seeding, proliferation, orientation, and differentiation of stem or progenitor cells on adhesive surfaces [Langer 1993]. DNA nanotechnology introduces a more ‘avant-garde’ approach to alter the biological behavior of cells to advance their morphology, viability, growth, and migration conditions, and accomplish pioneering bioengineering strategies at the cellular level.

Notable effects include the regeneration of cardiac tissue, bone and cartilage [Gonzalez-Fernandez, 2019], regulation of the nervous system for neural repair, regeneration and differentiation [Ma 2018], antiaging [Mao 2019], and anti-apoptosis effects [Shao 2018]. DNA-based nanostructures or nanomedicines can affect tissue regeneration and engineering through an excellently bioengineered capacity to prevent DNA disintegration prior to delivery and promote cellular internalization upon delivery in vivo.

Figure 5: A schematic representation of a logic-gated nanorobot designed and engineered with DNA origami for targeted transport of molecular payloads [Douglas 2012].

On a similar note, innovations can regulate DNA nanorobots or intelligent nanodevices with logic gates or simple on-off switches for cargo sorting, molecular computation, and biomedical diagnosis functions (figure 5) [Douglas 2012, Ma 2018]. Meanwhile, DNA nanohydrogels for biosensing roles can be engineered with dense packing of double-strand DNA instead of the classical Watson-Crick base pairing [Wang 2016].

Table 1: DNA-based nanostructures on tumor therapy and cell imaging [Ma 2021]. 

Future impact: bioinspired materials for DNA nanotechnology – bioengineering on a molecular playground.

From an investigational viewpoint DNA nanotechnology offers a wealth of potential applications for advances in bioengineering and biotechnology, with robust clinical outcomes already underway across tissue engineering research and cancer nanomedicine [Ma 2021]. The method branches out to a few interdisciplinary fields to cover tissue regeneration, cancer immunotherapy with vaccines, drug delivery and discovery, tumor therapy with nanorobots and implantable or injectable biomaterial-scaffolds with translational value (Figure 6). The technology offers a lab-on-a-molecule DNA nanoswitch platform to prompt DNA self-assembly and explore interactions of interest on a signature gel electrophoresis readout, to study molecular kinetics. Additionally, microRNAs are a fitting molecule for DNA nanotechnology based biosensing, with capacity to intercalate with molecular devices to accurately harness the complexity of DNA for functions beyond science fiction.

Figure 6: A molecular playground at the nanoscale. DNA-based nanostructures on preclinical bioimaging and diagnosis with an animal model with translational capacity on an organ chip instrument. The illustration shows how targeted ligands and cargoes can be attached to DNA-based nanostructures to enter target cells and release the DNA nanoarchitectures to selectively effect the region of disease [Ma 2021].

Despite the enormous promise of DNA nanotechnology, the method still has a few setbacks; diverse DNA nanomaterials can circulate poorly in blood, aggregate poorly in target tissues, or not arrive intact at the tissues or cells of interest. These shortcomings have led to investigations with other bionic materials such as liposomes and cell membranes to prolong blood circulation and the influence of DNA nanostructures; making room for materials scientists to investigate novel bioinspired materials for DNA nanotechnology. The structural flexibility, superior biocompatibility and multifunctionality of DNA nanotechnology provides a stimulating molecular playground for bioengineers to innovate strategies for DNA-based personalized nanomedicine.   

Header Image: An artist's representation of DNA nanotechnology. Image modified via credit: Wyss Institute for Biologically Inspired Engineering at Harvard.


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