HUH Tag Technology
HUH-endonucleases are diverse enzymes that break and join single-stranded DNA during biological processes such as rolling circle replication, rolling hairpin replication, bacterial conjugation, DNA transposition, and DNA integration into host genomes. During these processes, HUH-endonucleases cleave their substrate and form phospho-tyrosine covalent linkages to its newly exposed 5' end. Though these linkages are intermediated in their native contexts, they are capable of forming robust and non-labile linkages in vitro and in vivo. The lab has developed these nucleases as functional fusion partners, or 'HUH-tags', for sequence-directed protein-ssDNA bioconjugation.
Our lab was the first to solve the co-crystal structure of a Replication-Initiating HUH-Endonuclease in complex with its ssDNA substrate, revealing the complex network of inter- and intramolecular reactions that enable substrate binding and cleavage.
We are further pursuing the structures of other interesting or biomedically relevant members of this sub-family in complex with their cognate substrates.
Though our lab has interests in uncovering the molecular mechanisms of HUH-endonucleases and how they relate to their biology more broadly, our main interests are in applying them in biotechnology. We've had great success in implementing this bioconjugation tools as a means by which to directly tether a homology directed repair (HDR) template to Cas9, thus increasing the local concetration of repair donor at the site of a double stranded DNA break and enhancing HDR over lower fidelity means of repair, like the error-prone non-homologous end joining.
More recently, we've pursued these technologies in applications more pertinent to the lab's other goals through the development of 'RAD-TGTs', which are DNA-based tension probes that enable high-throughput characterization of the forces cells are able to output.
Engineering and Directed Evolution
A long standing goal of the Gordon lab is to uncover the molecular underpinnings of HUH-Endonuclease activity in order to modulate their specificity through rational engineering of 'designer' nucleases with unique specificity.
A more recent effort by the lab has been in developing high-throughput methods to direct the evolution of these nucleases towards non-cognate sequences and substrates -- stay tuned for updates in this area!
Current: Lidia Limón-Swanson, Adam Smiley, Matt Pawlak, Natalia Babilonia-Díaz
Alumni: Dr. Eric Aird, Dr. Kassidy Tompkins, Dr. Robert Evans