Effect of nanoscale, three dimensional arrangement of proteins on biological and therapeutic activity (FIT)

Slovene partner: UNG (Ario de Marco). Foreign partner: Jagiellonian University – Krakow (Jonathan Heddle)

UNG group members: Mirna Nakić, Matteo De March, Klara Kropivšek

Duration: Oct 2023 - Sept 2026

Topic:

The scope of this project is to design and manufacture nanoparticles using DNA and peptides. Such structures will have defined dimension and shape, with selective functionalization derivations at specific distances allowing the insertion of ligands. This concept will enable to define at the nm scale what ligands will be available and tune their interaction with their biological cognates. The scope is to obtain reagents that can bind simultaneously more independent epitopes of the targets. These can be virus or bacterial proteins involved in cell invasion or factors stimulating pathogenic processes.

It is a truism in protein biology that form determines function. This is clear for numerous protein-protein interactions including those of the immune system (e.g. antigen-antibody) and in in many examples of cell-pathogen interactions such as those between virus and host and between invasive pathogenic bacteria and target cells. It has become increasingly clear however that higher order levels of organisation are often vitally important for successful protein-protein (such as protein-receptor) interactions. Specifically, the three-dimensional arrangement of proteins as well as their density are now known to play a large role in determining successful interactions. Details of optimal arrangements are likely to vary depending on the specific molecules involved and to date only a limited number of systems have been comprehensively investigated. In our proposed work we will use designed protein and DNA nanoparticles to provide precisely controllable, three dimensional scaffolds onto which proteins can be attached and their spacing and density precisely controlled.

We will answer fundamental questions such as “What is the optimal spacings of proteins for the interactions? What is the optimal number of protein-protein interactions? What is the optimal density of interacting protein?” We have chosen to investigate two target systems using this approach: i) Modulation of SARS-CoV-2 binding capacity (with potential applications as an antiviral) and ii) Exploitation of the S. pyogenes invasion protein FbaB. This protein is used by S. pyogenes to invade the human endothelium through interactions with surface expressed integrins. We will investigate how FbaB displayed on designed nanoparticles can bind to receptors on endothelial cells (with potential applications in drug delivery) and how anti-FbaB binders exposed on artificial nanoparticles can inhibit S. pyogenes invasion of the endothelium (with potential applications as an antibacterial).

The Polish partner has been a world-leader in developing, designing and demonstrating both artificial protein cages and DNA cage nanoparticles with varying programmable shapes, sizes and geometries and having multiple attachment points to which peptides or folded proteins can be attached. He has also demonstrated the ability of such designed cages to carry protein cargoes in their lumen and deliver them to the interior of human cells. Finally, Heddle has considerable experience in determining cryo-EM structures of such protein and DNA particles and of protein complexes in general. At UNG we have experience in nanobody technologythat is instrumental for panning, selecting and producing binders for the functionalization of protein and DNA nanoparticles.

The project will involve in silico protein and DNA design. biochemical chemistry techniques for attaching displayed proteins to artificial nanoparticles in the correct manner, cryo-EM for determining and confirming the structures of selected complexes, fluorescent microscopy techniques for visualising nanoparticle-cell interactions and nanoparticle-mediated delivery of folded protein cargo to endothelial cells. The outcome of the project will provide fundamental insights into the role of three-dimensional arrangement in protein-protein interactions in nature as specific biophysical details of the particular interactions being investigated as well as significant translational potential acting as prototypes for further development as cell antibacterial and cell-delivery nanoparticles.

Project implementation:

Tasks 1.1 and 1.2 Adhiron library preparation and validation: the library has been synthetized and thoroughly tested with a large array of antigens. Task 2.1 Antigen preparation: the protocols for RDB and FbaB production have been established and enough material recovered for panning and binder evaluation. Nevertheless, the project will probably require some further purifications in the late stages for the characterization of the functionalized nanoparticles. Tasks 2.2 and 2.3. Adhiron and nanobody isolation; these tasks are virtually completed, but we consider the possibility to make a further panning whether the performance of the already available binders would be not corresponding to the expectations. Task 2.4 Binder production: we already produced a huge number of constructs, but there is a constant request from our partner to modify some sequences and tag combinations to obtain reagents that might better perform on the nanostructures. Task 2.5. Biophysical characterization and optimization of the binder features: this is the last step in the pipeline and, consequently, it is the one that will require more commitment in the last phase of the project. Whereas the biophysical characterization of the binders has well advanced, the modeling and docking optimization is just at the beginning. Finally, we got crystals of some binder/antigen complexes, but they did not refract enough and a new attempt has started.

Publications:

D’Ercole C, De March M, Veggiani G, Oloketuyi S, Svigelj R, de Marco A (2023) Biological applications of synthetic binders isolated from a conceptually new Adhiron library. Biomolecules 13:1533 COBISS.SI-ID 168816899

D’Ercole C, de Marco A (2023) Native agarose gels and contact blotting as means to optimize the protocols for the formation of antigen–ligand complexes. Bioengineering 10:1111 COBISS.SI-ID 165464323

Neumair J, D’Ercole C, De March M, Elsner M, Seidel M, de Marco A (2023) Macroporous epoxy-based monoliths functionalized with anti-CD63nanobodies for effective isolation of extracellular vesicles in urine. Int J Mol Sci 24:6131 COBISS.SI-ID 146615299

Li J, Kang G, Wang J, Yuan H, Wu Y, Meng S, Wang P, Zhang M, Wang Y, Feng Y, Huang H, de Marco A (2023) Affinity maturation of antibody fragments: A review encompassing the development from random approaches to computational rational optimization. Int J Biol Macromol 247:125733 COBISS.SI-ID 159012099

Wang J, Kang G, Lu H, de Marco A, Yuan H, Feng Z, Gao M, Wang X, Wang H, Zhang X, Wang Y, Zhang M, Wang P, Feng Y, Liu Z, Cao X, Huang H (2024) Novel Bispecific Nanobody Mitigates Experimental Intestinal Inflammation in Mice by Targeting TNF-α and IL-23p19 Bioactivities. Clinic Translat Med 14:e1636 COBISS.SI-ID 190583043

Štrancar A, D’Ercole C, Nakić M, Cikatricisova L, De March M, de Marco A (2024) A practical guide for the quality evaluation of fluobodies/chromobodies. Biomolecules 14:587 COBISS.SI-ID 195579651

de Marco A (2025) Recent advances in recombinant production of soluble proteins in E. coli. Microb Cell Factories 24:21 COBISS.SI-ID 222590211