Gerasimaite, R.; Bucevicius, J.; Bubnyte, D.; Koenen, T.; Lukinavicius, G.

A comprehensive understanding of protein-nucleic acid interactions in the crowded nuclear milieu is essential for elucidating genome function. State-of-the art methods provide the finest details of genome organization, but lack integration of imaging and sequencing modalities. We introduce OptiFoot, a fluorescence-based platform for recording protein-nucleic acid interactions in cells. OptiFoot employs an engineered sequence-unspecific N6-adenine methyltransferase that attaches diverse functional groups to DNA and RNA. When targeted to protein of interest by genetic fusion or antibody, it generates covalent high contrast fluorescent footprints that can be visualized by super-resolution microscopy and analyzed by optical mapping of single native DNA molecules, allowing complementary spatial and genomic analyses. To illustrate OptiFoot versatility, we imaged lamina-associated domains, DNA replication sites, CTCF binding sites and histone modifications in human cells and produced corresponding genome profiles. We use OptiFoot to demonstrate that majority of nucleoporin NUP153-DNA interactions occur in nucleoplasm, outside nuclear pore.