In a cell nucleus DNA is organized in a complex 3D multiscale architecture. This architecture plays a role in DNA functions, such as gene regulation, but is not fully understood (Bickmore 2013)(Hnisz et al. 2016)(Lupiáñez et al. 2015). New techniques to study the dynamics of DNA in single living cells would be useful to better understand this architecture. In this project we propose to build magnetic nanoprobes that can be attached to DNA. They are iron oxide magnetic nanoparticles and will be used to apply a mechanical force on a specific part of the genome. This could give a precise insight on how the genome is sensing a mechanical force and what is its effect on transcription.

The first challenge was to avoid nonspecific interactions between nanoparticles and biomolecules in the very crowded nucleus. To achieve this we designed a specific diblock polymer to coat the nanoparticles. The first block is made of a poly(sulfobetaine) zwiterrion which ensures colloidal stability in cells and has interesting antifouling properties. The second block is a poly(phosphonic acid) which ensures the anchoring stability of the polymer to the nanoparticle on long time scales. The absence of non specific interactions with the polymer ligands has been assessed in vitro and in cells by fluorescence correlation spectroscopy. We show that the nanoparticles do not interact with model albumin proteins and components of the serum. They diffuse freely in the cytoplasm and the nucleus of living cells.

The second challenge is to bind these nanoparticles to specific DNA sequence. This has been achieved by conjugating a GFP by click chemistry on the surface ligands of the nanoparticles. In the nucleus, this GFP is recognized by an anti-GFP nanobody which is bound to a tetR protein. This tetR protein binds in turn to the repeated gene sequence tetO in our model cell. Thanks to this magnetic nanoprobe bound to DNA it will be possible to pull on DNA with a magnetic micro array and to study DNA architecture.

Bickmore, Wendy A. 2013. “The Spatial Organization of the Human Genome.” /Annual Review of Genomics and Human Genetics/ 14(1): 67–84.

Hnisz, Denes et al. 2016. “Activation of Proto-Oncogenes by Disruption of Chromosome Neighborhoods.” /Science/351(6280): 1454–58z

Lupiáñez, Darío G. et al. 2015. “Disruptions of Topological Chromatin Domains Cause Pathogenic Rewiring of Gene-Enhancer Interactions.” /Cell/ 161(5): 1012–25.