Self-assembling aptamer-protein hybrid systems binding biologically relevant ligands

Self-assembling aptamer-protein hybrid systems binding biologically relevant ligands

Sekcia: 
Rok:
2023

Celkové hodnotenie

Vedecká práca
75%
Prevedenie (dizajn)
75%
Diskusná interakcia
75%
PoužívateľVedecká prácaDizajnDiskusná interakcia
MUDr. Dominik Michálek100%100%100%
RNDr. Monika Švecová100%100%100%

Self-assembling aptamer-protein hybrid systems binding biologically relevant ligands

Viktoria Fedorova1 , Tomas Biro2 , Katarina Siposova , Martin Humenik3 ,
1 Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice, Slovakia
2 Department of Biophysics, Institute of Physics, P. J. Safarik University in Kosice, Kosice, Slovakia
3 Department of Biomaterials, Faculty of Engineering Science, Universität Bayreuth, Bayreuth, Germany
fedorova@saske.sk

Protein amyloid structures are well known as factors causing  diseases called amyloidoses, such as Alzheimer’s or Parkinson’s disease. However, a group of „functional amyloids“ has been identified which are fulfilling natural purposes in mostly lower eukaryotes, for example bacteria, fungi or spiders1. Amyloids possess characteristic structural cross-β structure consisting of the β-sheets arranged perpendicularly along the fibril axis. Among the proteins which self-assemble into the fibrils, recombinant spider silk proteins have shown high potential for the use in biomedical as well as technological applications based on advantageous properties of the processed materials such as nanofibrils, particles, coatings, foams, fibers or hydrogels, showing biocompatibility, biodegradability and the absence of immune response of the living organisms2. The purpose of this study was to prepare DNA-spider silk protein bioconjugates using recombinant spider protein eADF4(C16), which is derived from the natural dragline protein of the European garden spider Araneus diadematus fibroin 4 (ADF4). The recombinant protein provides the possibility of site-specific chemical modification with oligonucleotides3,4. We used aptameric nucleic acids TBA15 and TBA29 which are known for binding of human thrombin. For the synthesis of these bioconjugates, the catalyst-free „click”-chemistry reaction was used after the modification of the protein N-terminus with azide (N3) and the 5´-end of the oligonucelotides with dibenzocyclooctyne (DBCO). The accuracy of initial modifications and subsequent „click”-conjugation was verified using methods such as high-performance liquid chromatography (HPLC), mass spectrometry (MALDI-TOF) and native PAGE electrophoresis. Subsequently, our aim was to characterize the TBA15(29)-eADF4(C16) bioconjugates in terms of their ability to self-assembly kinetics in the presence of different ions using circular dichroism (CD) and atomic force microscopy (AFM). The experiments were carried out using different concentrations of phosphate ions (Pi), which is proved to promote fibril formation of the spider silk protein2. The next step was various concentrations of Na+ and K+ ions to determine their impact on the aptamer secondary structure and the rate of the bioconjugate self-assembly. The data suggest that the bioconjugates TBA15(29)-eADF4(C16) retain their ability to self-assembly into higher structures while maintaining the same properties as naturally occurring protein cross-βfibrils. The kinetics of the self-assembly process appeared to be highly affected by the phosphate ions in the solution. Initial measurements of the kinetics at different conditions suggested that there was a more significant impact on the self-assembly caused by the Na+ ions in the solution than the K+ ions. Our observations indicate that there is a great potential for using the spider silk protein-DNA bioconjugates to prepare highly organized structures with the ability to bind biologically relevant agents, such as thrombin which could lead to the practical applications, for example as the drug delivery systems or biosensors.

Poďakovanie: 

This work was supported by the grants: PPP SK-DAAD and VEGA 2/0034/22.

Zdroje: 

1 Iadanza, M.G., Jackson, M.P., Hewitt, E.W., Ranson, N.A., Radford, S.E., 2018. A new era for understanding amyloid structures and disease. Nat Rev Mol Cell Biol 19, 755–773. https://doi.org/10.1038/s41580-018-0060-8

2 Humenik, M., Preiß, T., Gödrich, S., Papastavrou, G., Scheibel, T., 2020. Functionalized DNA-spider silk nanohydrogels for controlled protein binding and release. Mater Today Bio 6, 100045. https://doi.org/10.1016/j.mtbio.2020.100045

3 Humenik, M., Drechsler, M., Scheibel, T., 2014. Controlled hierarchical assembly of spider silk-DNA chimeras into ribbons and raft-like morphologies. Nano Lett 14, 3999–4004. https://doi.org/10.1021/nl501412k

4 Humenik, M., Scheibel, T., 2014. Nanomaterial building blocks based on spider silk-oligonucleotide conjugates. ACS Nano 8, 1342–1349. https://doi.org/10.1021/nn404916f

Diskusia

Ad: "Batch-to-batch" differences

Congratulations, a nice piece of work, state-of-the-art experimental tools and techniques. I am curious about how does the random nature of the self assembly influence the resulting bioconjugate - its desirable final structure-function relationships. Based on your experimental experience, do the bioconjugates prepared under the same experimental conditions provide consistent results (behaviour)? Thank you.

iwa

Thank you for your reaction and for the questions.

According to our initial measurements, the structure of bioconjugates, in comparison with structure of recombinant spider silk protein eADF4(C16), shows only a small, insignificant differences when there is no ligand in the solution. The most visible difference in the self-assembly is in its rate which is  influenced by various concentrations of ions that incite the self-assembly process and it's seen for the pure recombinant protein and bioconjugates as well. However, initial experiments using the mix of bioconjugates in solution with relevant ligand, e.g. thrombin, show that since different aptamers used (TBA15 and TBA29) bind to different binding sites of thrombin, these fibrils are then capable of binding among themselves and form higher organised raft-like structures with potential functional improvements when compared with pure recombinant protein/bioconjugate.
On the question of the consistency of the results – so far our experiments show high reproducibility and consistent results, when the same experimental conditions are applied. 

Viktória

Thank you for your reply and great work on this project. Can you be a little bit more specific about your contribution to this work?

iwa

Thank you for your kind words.

This project is a collaboration between our research group in Košice and research group of Dr. Humenik at Bayreuth University in Germany – that is also a place where I prepared the bioconjugates used, starting with modification of spider protein and oligonucleotides under direct supervision of Dr. Humenik and verification of these reactions as well. At our Institute of experimental physics in Košice, I performed the kinetics measurements and their evaluation under the guidance of my PhD supervisor, Dr. Šipošová. At the same time, for this project I act as a consultant for our diploma student, Tomáš Bíró, who was also a part of preparing the kinetics measurements done and shown in this poster.

Viktória

Thank you.

I wish you success in all your future endeavors.

iwa

Thank you very much! :)

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