DNA Nanodevices

The MEOS Innovation Center for Precision Analysis of Cell Therapy Products (abbreviated: MIC-PreCell) is currently being established at the Fraunhofer Center for Microelectronic and Optical Systems for Biomedicine (MEOS) in Erfurt. 

This involves developing new analysis methods for quality assurance and process control for the production of cell-based therapeutic products. As part of the project, infrastructure and know-how are combined to close the current gap between technologies which are available in principle to analyze cell-specific parameters and their broad application in the production of cell-based medication. 

Usually, cell-based treatments are tailored to the specific patient and, due to the extremely complex production processes, they are often also very expensive. In addition, very rapid production is often essential for the survival of patients in a critical phase of the disease. Therefore, as part of the project, modern methods of integrated quality assurance are to be established to shorten production processes and identify potential production errors much earlier. In this context, the focus is on the broad use of innovative quality assurance methods in cell production, such as optomechanical profiling, with the help of which mechanical cell properties can be determined without labeling immediately. Moreover, VOCs (volatile organic compounds) which are emitted by cell cultures into the air are to be analyzed with the help of a gas chromatograph ion mobility spectrometer. In addition, equipment for the micromanipulation of cells and cell clusters or organoids will be used, permitting the acquisition of direct and real-time information on the state of therapeutic cell products.

Many standard medical diagnostic methods are time consuming and, moreover, they do not take account of the individual differences between patients. This can lead to incorrect or incomplete diagnoses and sub-optimal treatment decisions. 

At the Fraunhofer Center for Microelectronic and Optical Systems for Biomedicine (MEOS), researchers of Fraunhofer IZI, IPMS and IOF are jointly developing disposable biosensors with the aim of improving the analysis speed, the number of measurement parameters and the precision of the results.

The photonic biosensor chips are developed on a silicon nitride waveguide platform at the Fraunhofer IPMS. These biosensors consist of specifically developed, scalable micro-ring resonators with several channels. The detection method is based on specialized bioassays developed at Fraunhofer IZI. In this process, specific catcher modules are bound to functionalized sensor layers whose transmission spectrums change as soon as corresponding analytes bind to the catcher molecules. These biosensors are highly sensitive and are suitable for detecting biomolecules in bodily fluids - which makes it valuable for the early detection of illnesses. 

The research team has successfully developed a demonstrator based on a multi-channel silicon nitride micro-ring resonator biosensor system. This system permits the multiplex detection of specific miRNA biomarkers connected with neurodegenerative diseases. They are detected with the help of DNA-based catcher molecules immobilized on the sensor surface. The developed sensors and the integrated system are versatile and can be adapted to detect various biomarkers, viruses or bacteria in different fluids.

Partners
Fraunhofer Institute for Photonic Microsystems IPMS; Fraunhofer Institute for Applied Optics and Precision Engineering IOF

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. The approach allows precise arrangements of defined glycan chains with single-nanometer spatial resolution to be established. This brings together two key technologies from the partners Fraunhofer IZI and the Max Planck Institute for Colloids and Interfaces: DNA Nanotechnology and Automated Glycan Synthesis.

One part of this project focuses on creating high-throughput assays for investigating the binding of specific glycan formulations and arrangements to target pathogens or glycan-binding proteins. For this, glycosylated DNA nanostructures have been integrated into two standard, analytical platforms that are widely accessible to researchers around the world.

First, 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. Alternatively, we different types of DNA-glycan nanostructures with the hardware used for carrying out classical ELISA assays. Thus, synthetic replacements for antibodies used to capture and detect in immune-diagnostics were created. Unlike standard ELISAs or other similar assays, this method allows rapidly screening through many candidate ligands, and additionally controlling the geometric arrangement in which they are presented to targets proteins.

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.

• Development of molecular carrier and immunological systems from DNA and hybrid materials
• Energy conversion and ordering phenomena in DNA-based and composite nanomaterials
• Mechanical characterization of DNA-based and composite nanomaterials