Visualizing looping of two endogenous genomic loci using synthetic zinc-finger proteins with anti-FLAG and anti-HA frankenbodies in living cells

Abstract

In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here we demonstrate a new method to visualize endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps. First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously visualize the dynamics of the two loci in single living cells. This reveals close association between the two loci in the majority of cells, but the loci markedly separate upon the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof-of-principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.

Single-molecule visualization of mRNA circularization during translation

“…Nevertheless, it is still difficult to track single or multiple translation regulators at the same time owing to the high intracellular concentration of the targets 74,75 , although one example has been reported 50 . Further advances in protein labeling strategies are also required beyond the several methods that have been developed, including small peptide tags and their cognate antibodies [82][83][84] , the incorporation of unnatural amino acids 85 chemical labeling without genetic manipulation 86 . Because of its high specificity and efficiency, the Halo-87 or 88 is still the preferred strategy despite the potential problems caused by the large tag size.…”

NanoTag Nanobody Tools for Drosophila In Vitro and In Vivo Studies

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