Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering

摘要: Abstract Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are basis for HUH-tag bioconjugation a critical adeno-associated virus genome integration tool. We solved first co-crystal structures of complexed ssDNA, revealing key motif conferring sequence specificity anchoring bent architecture. combination, we developed deep sequencing cleavage assay, termed HUH-seq, interrogate subtleties Rep demonstrate how differences can be exploited multiplexed HUH-tagging. Together, our insights allowed engineering only four amino acids chimera predictably alter specificity. These results have important implications modulating viral infections, developing Rep-based genomic tools, enabling massively parallel barcoding applications.