DNA Origami-based machines for epigenetic manipulation of gene transcription – ChromOrigami

Investigating epigenetic and related transcriptional changes is key to understand how transcription is regulated in the native cellular environment. At present many of the results of mapping of chromatin modifications or gene expression measurements use indirect methods. We have engineered DNA origami (DO) nanoscale machines that can target specific biomolecules, emit light, detect nucleic acids or other molecules of interest, and apply external forces in vitro. Recently, in a collaborative effort we demonstrated that functionalized DOs can be targeted to the nuclei of human cells, which also established critical experiment workflows within our consortium, composed by two US- and two France-based labs. In this project we will build on our previous NSF founded collaboration, and leverage these DO nanodevices to directly manipulate transcriptional regulation of target genes. Our efforts incorporate in a long-term aim of using DO nanodevices to regulate and/or visualize transcription in live human cells. This project plans to develop a set of tools that can be used to monitor and control transcription in live human cells. These tools include: (i) multi-functional DOs to target, visualize gene loci and activate transcription of specific target genes; (ii) DOs that detect and measure transcription from either a single gene or multiple genes;(iii) reconfigurable and multifunctional DOs that can carry out functions in response to external or endogenous cues within cells, such as inducible nuclear localization and/or transcriptional activation. The detection read-out of these multifunctional DOs will be then analysed by a user-friendly computational platform combining advanced deep learning and biophysical modelling to perform automatic imaging analyses and estimation of fundamental kinetic rates of diffusion and transcription. We will use and combine the cutting-edge and the state-of-the-art expertise of the four labs in single molecule biophysics, DNA origami nanotechnology, molecular biology, cell biology, advanced imaging and deep learning to determine the DO-based influence of direct targeting and manipulations on the dynamics of transcription regulation and chromatin environment. Overall, these studies will provide the first direct evidence of how changes in gene regulation and/or chromatin compaction influence transcription activator binding, co-activators function and transcription regulation in live cells. In addition, our planned project will establish the manipulation of nanodevices that can be used to influence gene expression in live cells and consequently can be further developed to be used in complex biological systems, such as in diseases.