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Molecular Analytical Robotics Assays

Periodic Reporting for period 3 - MARA (Molecular Analytical Robotics Assays)

Okres sprawozdawczy: 2018-06-01 do 2020-08-31

Diagnostic tests are essential to provide a targeted treatment of infectious diseases and to prevent the further spread of multidrug resistant pathogens. Current methods are either cultivation- or PCR-based and therefore entail significant limitations concerning the clinical need and requirements to characterise pathogens including their resistance mechanisms within 3 hours. In MARA, we aimed to develop and combine three radically novel technologies that should lead to substantial breakthroughs in science, medicine and industry and, as proof-of principle, use them to create a DNA-based molecular toolkit for the characterisation of pathogens.
First, for the detection of pathogen-associated antigens we developed Autonomous Detection Nucleic Acids (AUDENA) that are independent of any laboratory instruments and sophisticated processing. The realisation of the AUDENA concept could lead to an autonomous, stable, simple and very economic novel sensor class applicable for any water-soluble substances. The AUDENAs are superior sensors that can be used for almost all chemical substances and require just a translucent container and a person to identify the colour change. Costs in mass production are below 1€, and thus, AUDENAs could replace current diagnostic and environmental tests.
The second revolutionary technology in MARA employs a novel approach in protein mimicry and creation of artificial enzymes, which represents a breakthrough in several disciplines, such as biotechnology, biomedical manufacturing and the energy sector. A so-called DNA Scaffold Embedded Protein Emulation Complex (D-SEPEC) was created to integrate amino acid-made catalytic centres into supramolecular DNA origami structures and in order to emulate the biological rotary motor of the archaellum and an ion channel using DNA strands.
The third targeted breakthrough in this project was the development of a molecular drill that specifically identifies target cells and destroys them by altering their osmotic system. It was planned to realise this Molecular Robot (MORO) as a DNA nanostructure, driven by an ATP-powered D-SEPEC and aptamers for target recognition. The MORO should lyse the bacterial cells in order to release intracellular antibiotic resistance associated antigens. However, the long-term vision anticipates an application as antibiotic replacement for infectious diseases.
Apart from DNA nanodevice applications in industrial processes, their potential for medical applications such as cancer therapeutics and surgical interventions to counter e.g. artery blockage is tremendous. Being not only highly specific but also biodegradable, the MOROs could have almost unlimited potential in medicine, biotechnology and science. Recent studies have demonstrated the targeted lysis of tumours in mice using DNA nanostructures functionalised with aptamers and proteins.
During the MARA project, the realised actions were focusing on the development of the AUDENAs and the D-SEPEC as prerequisites for the MOROs. First, functional AUDENA prototypes were designed as a proof-of-concept using published DNA aptamers as target recognition element. They consist of an aptamer domain and a DNAzyme, in which the DNAzyme activity can be regulated by the aptamer-target interaction. The complex of G-quadruplex (GQ) and hemin is a peroxidase-mimicking DNAzyme and has become increasingly popular as a reporter system for biosensing applications. The development of GQ-based AUDENAs is of high interest as they can be used as label-free biosensors for the direct and real-time detection of pathogens. Herein, we rationally designed ca. 400 GQ-based AUDENA candidates and evaluated the suitability of 25 aptamers targeting seven variant proteins and small molecules for this detection concept. As a result, six novel AUDENA were developed for the specific detection of quinine. In addition, a systematic approach was developed towards the rational design of AUDENA. Using an enzyme-linked oligonucleotide assay (ELONA) and fluorescence Thioflavin assay, we performed experiments to find how the affinity of aptamers is affected once conjugated to the DNAzyme sequence or upon integration into the AUDENA probe. Furthermore, we investigated the impact of the structure-switching functionality in the parent aptamer and the effect of the reaction matrix on the efficiency of probes.
The realised actions concerning the development of the D-SEPEC included the expression and purification of the archaellum and ion channel. First, cryo tomography experiments were conducted to analyse the archaellum and its function. Significant progress was achieved in understanding the function of the archaellum. In parallel, the software Adenita was developed to facilitate the design of the D-SEPECs. Furthermore, molecular dynamics simulations of the archaellum were calculated. Based on these results, a D-SEPEC was designed and its functionality evaluated.
In parallel, the scaffold structure of the final nanodrill was designed, and a technical framework to analyse the nanomechanical rotational behaviour of such a small device was developed. It was finally functionalised with an aptamer targeting Staphylococcus aureus. The successful binding of the DNA nanostructure to the bacterial pathogen was confirmed by cryo electron microscopy.
The highly ambitious MARA project has achieved some scientific progress beyond the state of the art. In general, the DNA nanostructure research field is highly dynamic and significant high-impact results are published constantly such as a DNA nanostructure that can specifically destroy tumours in vivo. Thus, a reliable forecast on the long-term impact of this project is difficult to provide. However, we consider that the AUDENAs, the nanomodelling software Adenita, the new knowledge about the archaellum and the D-SEPEC concept represents a significant progress in the field of DNA nano and biotechnology.
Cell detection and lysis