Polysaccharides, also known as glycans, are long and complex sugar molecules made up of a chain of monosaccharides such as mannose, glucose or fructose. Numerous glycans are found on the surface of human cells, and are used by disease-causing bacteria or viruses as molecules for recognizing, binding, and eventually infecting the host cells. Therefore, sugar molecules such as mannose, heparin or sialic acid, which are found on the membrane of human cells, are particularly interesting from a medical research perspective. Nanometer-scale geometry also plays an important role here; viruses and bacteria exploit the principles of multivalence, where two or three sugar-binding receptors act cooperatively to more efficiently bind to and infect their targets.
In Glyco3Display, novel carbohydrate-based compounds are created by integrating different glycan molecules with DNA-based structural scaffolds. This approach leads to precise arrangements of defined glycan chains being created with single-nanometer spatial resolution. This brings together two key technologies from partners Fraunhofer IZI and the Max Planck Institute for Colloids and Interfaces: DNA Nanotechnology and Automated Glycan Synthesis.
In the early phases of the project, work focused on creating a high-throughput assay for investigating the binding of specific glycan formulations and arrangements to target pathogens like E. coli bacteria. By integrating the DNA-glycan compounds onto microbeads, any standard automated flow cytometry system can be used to quantify the impact that the exact glycan composition and how they are geometrically arranged on DNA scaffolds has on their ability to bind the surface of the pathogen.
Using the information gained from this assay system, new DNA-glycan compounds can be designed for both diagnostic and therapeutic purposes. By covering the surface of viruses or bacteria with these compounds, their ability to infect host cells can be significantly hindered. These types of “anti-adhesive” or “fusion inhibition” compounds are increasingly being developed for medical markets. Similarly, the specific binding of these DNA-glycan compounds to the surface of pathogens can be utilized instead of traditional capture antibodies in advanced disease diagnostics.
The field of DNA nanotechnology utilizes DNA strands not for their genetic encoding capabilities, but rather as construction materials. Using rational design principles, individual DNA strands can be assembled into precise nanostructures of nearly any shape. These nanostructures allow functional molecules such as peptides to be attached to nearly any unique location on their structure. Since structural features can be altered with the spatial resolution of a single base pair (0.34 nanometers), several molecules can be attached in closely controlled geometry. When these molecules are ligands that bind to specific targets, their spatial arrangement can be controlled according to the desired target’s geometry. This results in optimized binding and/or signaling interactions.
In this project, the efficacy of SWL, an ephrin-mimicking peptide that binds specifically to EphrinA2 (EphA2) receptors, was enhanced by a factor of nearly four orders of magnitude by presenting three of these peptides on small DNA nanostructures in an oligovalent manner. Ephrin signaling pathways are critical in the development and progression of many types of cancer, and are potential targets in cancer diagnosis, imaging and treatment.
Here, the impact of SWL valency on binding affinity, phosphorylation (a key player for activation) and the regulation of phenotype prostate cancer cells that express EphA2 was quantitatively demonstrated. DNA structures with three SWL peptides significantly enhanced EphA2 phosphorylation by 8000-fold. Furthermore, the pinpointed interaction of these constructs showed an enhanced impact on the retraction of cells compared to one of EphA2’s natural ligands – ephrin-A1. These results demonstrated that simple DNA structures can be used to greatly enhance the potency of otherwise weak signaling peptides, using principles of a nanometer-scale oligovalent arrangement.
Beyond its standard role as the carrier of genetic information in living organisms, DNA has also emerged has highly versatile construction material for fabricating nanometer-sized particles and machines. By carefully designing the sequences of DNA strand collections, complementary base pairing can be used to fully control the size, shape and mechanical properties of single, DNA-based nanoparticles or larger DNA-based materials.
An example includes materials formed from DNA nanotubes. A small collection of DNA strands is designed to self-assemble into micrometer-length filaments. Their nanometer-sized diameters can be precisely controlled in order to "program" their nanoscale mechanical properties. These can act as synthetic mimics of biologically derived structures such as actin or collagen filaments. However, the programmable nature of DNA strands provides the ability to selectively control parameters such as the stiffness of the individual nanotubes, which is not possible with biologically derived materials such as actin or collagen.
By forming the DNA nanotubes in a crowded molecular environment, similar to what is found inside cells, they can be made to spontaneously assemble large microstructures that are dependent upon their stiffness and volume fraction (Figure 1). These star-like or bundled structures resemble cellular structures such as stress fibers, filopodia or the mitotic spindle, and are tools to provide insight into the basic mechanism behind their formation in biological systems.
Additionally, DNA nanotubes at lower volume fractions form entangled, elastic hydrogels. Here, the elastic plateau shear modulus (G') can be adjusted over a wide range by changing either the network density or the stiffness of the individual DNA nanotubes. This enables a fine-tuning of the hydrogel stiffness while being able to independently maintain factors such as porosity. This ability to control macroscale properties through programmable nanoscale building blocks can be applied more broadly to develop functional materials for cell-based applications such as 3D cell culture or nutrient repositories in long-term bioreactors.