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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MARA (Molecular Analytical Robotics Assays)

Teaser

Summary

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 will develop and combine three radically novel technologies that will 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, the detection of pathogen-associated antigens will be performed by Autonomous Detection Nucleic Acids (AUDENA) that are independent of any laboratory instruments and sophisticated processing. The realisation of the AUDENA concept will 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 1â‚¬, and thus, AUDENAs could replace almost all diagnostic and environmental tests within the next fifteen years.
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) will be created to integrate amino acid-made catalytic centres into supramolecular DNA origami structures and to emulate the mechanical motion of a biological rotary motor and an ion channel using DNA strands.
The third breakthrough in this project is the development of a molecular drill that can specifically identify target cells and destroy them by altering their osmotic system. This Molecular Robot (MORO) will be made of DNA, ATP powered D-SEPECs and Seligos, which are aptamer-like structures consisting of DNA and amino acids. In MARA, the MORO will be used for the lysis of bacterial cells to release intracellular antibiotic resistance associated antigens. However, the long-term vision anticipates an application as antibiotic replacement for infectious diseases and a therapeutic agent for cancer treatment, which would represent one of the most important breakthroughs in medicine in the recent years. Using specially designed, artificial molecular machines for a highly targeted attack on pathogens or tumour cells may well cause a paradigm shift in our approach to disease therapy and open up a whole new area in molecular medicine.
Apart from the MOROsâ€™ application in industrial processes, their potential for medical applications such as surgical interventions to counter e.g. artery blockage is tremendous. The coupling of the machines with target-recognising elements enables the design of various, specialised MOROs. In addition, the long-term vision of our innovative technological approach to emulate any protein activity would allow applications in the energy sector. Light-harvesting D-SEPECs inspired from plants or bacteria could be used for the generation of an electron flux in electronics or the generation of clean fuels such as H2. Other envisioned applications involve synthetic processes where cascades of different D-SEPECs can produce any sophisticated material available in nature. The programmability of the catalytic reactions and their arrangement in space would allow the creation of micro- and macroscopically definable materials made of cotton, wood, wool, leather, etc. Realistically, massive resources and time will have to be invested to reach these sketched long-term visions if the D-SEPEC concept is successful.
Being not only highly specific but also biodegradable, the MOROs have almost unlimit

Work performed

In the first 12 months of the project, the realised actions were focusing on the development of the AUDENAs and the D-SEPEC. A first functional AUDENA prototype was designed as a proof-of-concept using a published DNA aptamer instead of a Seligo as target recognition element. The sample comprising hepatitis B e antigen could be clearly differentiated from the sample lacking an antigen. In parallel, the first Seligos targeting the clinically important antibiotic resistance protein OXA-48 were developed. Based on these Seligos, various AUDENA designs were developed and are currently being synthetized.
The realised actions concerning the development of the D-SEPEC include the expression and purification of the archaellum and ion channel. First cryo tomography experiments were conducted to analyse the archaellum. In parallel, the software development to facilitate the design of D-SEPECs has started and molecular dynamics simulations of monomers of the archaellum were calculated. In addition, first DNA nanostructures mimicking structural components of the archaellum were designed and visualised by electron microscopy.

Final results

The highly ambitious MARA project is after 12 months still at a very early stage. The DNA nanostructure research field is highly dynamic and significant high-impact results are published constantly. Thus, a reliable forecast on the progress beyond the state of the art and expected potential impact is difficult to provide. However, we consider that the D-SEPEC concept developed and refined in the first 12 months represents a significant progress in the field of artificial enzyme design. In addition, the potential application of Seligos as inhibitors of antibiotic resistance would represent a progress beyond the expected potential impact.