Development of a DNA origami nanosensing platform for diagnostic analysis of ADAMTS-13 proteolytic activity

• Applicants: Dr. Viktorija Glembockyte, Ph.D.; Dr. Susanne Weber
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry ; Biochemistry ; Toxicology, Laboratory Medicine
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 503042693

The DNA origami technology provides a powerful tool for the design of a wide range of static and dynamic nanostructures. The programmable nature of DNA base pairing allows the generation of clearly defined structures, which can be applied in numerous applications, ranging from biosensing and drug delivery to development of DNA-based nanorobots. The proposed project aims to establish a DNA origami-based nanosensor platform for the analysis of the protease A Disintegrin And Metalloprotease with ThromboSpondin-1-like domains 13 (ADAMTS-13) in human plasma. Dysfunctional or absent ADAMTS-13, which physiologically cleaves ultra-large von Willebrand factor multimers in the bloodstream, causes the disease thrombotic thrombocytopenic purpura (TTP), a severe and life-threatening thrombotic microangiopathy (TMA). Analysis of ADAMTS-13 activity as well as quantification of antigen and autoantibodies to the antigen are crucial for the diagnosis of TTP and its differentiation from other TMAs. Currently the analysis of ADAMTS-13 is often restricted to specialized laboratories. In order to accelerate the differential diagnosis and optimal treatment of TTP and other TMAs, we aim to develop a sensitive and point-of-care compatible sensing platform by leveraging the modular nature of DNA origami nanotechnology to completely decouple biorecognition and signal transduction elements of the biosensor. The envisioned biosensors are based on dynamic DNA origami hinge-like nanostructures conjugated to ADAMTS-13 specific polypeptides (either ADAMTS-13 target peptide or ADAMTS-13 epitope). The DNA origami hinges are driven towards the open state due to electrostatic repulsion between the negatively charged hinge arms, but can be closed with the help of hybridizing DNA strands or bridging polypeptides. Fluorophore pairs properly placed on the hinge arms and allowing fluorescence resonance energy transfer (FRET) in the closed hinge state will serve as signal transduction element. Whereas, protruding DNA strands at the end of the hinge arms, which will be modularly linked by complementary DNA functionalized protease target peptides or other interaction partners, serve as biorecognition elements. Cleavage of the bridging peptides by targeted proteases or replacement of interaction partners will lead to an opening of the hinge-like nanostructure resulting in a loss of the FRET signal. Thus, the biosensor platform will be able to report directly on the activity and/or concentration of ADAMTS-13 in blood samples.The key advantage of the proposed biosensor design is a complete decoupling of biorecognition and signal transduction elements enabling the detection of highly sensitive targets, such as ADAMTS-13, without compromising the signal response of the biosensor. Due to the modular design of the projected nanosensors, the sensing platform can also be easily adapted for the analysis of other relevant proteases or nucleases.

• DFG Programme: Research Grants