Today we report the directed evolution of HUH into rHUH, a sequence-specific covalent protein tag for RNA labeling. rHUH was hard to engineer: the initial HUH template had no RNA activity, so we started with RNA-DNA hybrids. 30 rounds of selection on the yeast surface were required, and 7 cycles of
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The final rHUH tag is 13.4 kD, has 12 mutations relative to HUH, and covalently labels as little as 1 nM target RNA within minutes. The labeling works well in mammalian cell lysate, but not yet in the interior of living cells – possibly due to the high Mg2+ requirement
There is still work to do for this to be a dream tool for RNA imaging/editing/mapping in living cells, but rHUH provides a good starting point and it is definitely good enough for in vitro
Congratulations to postdoc Rongbing Huang, whose creativity, persistence, and resourcefulness made rHUH possible! We are grateful to NSF and Biohub for funding this work.
https://www.pnas.org/doi/10.1073/pnas.2422085122