HUH Endonuclease: A Sequence-specific Fusion Protein Tag for Precise DNA-Protein Conjugation

Du, Jiajun; Kong, Yuhan; Wen, Yujian; Shen, Enxi; Xing, Hang

doi:10.1016/j.bioorg.2024.107118

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A self-catalyzing strategy for co-immobilization of two distinct proteins at equimolar ratio: A case study of 3A and 2C to develop a chromatographic method for finding prospective dual-target compoundsfrom complex matrices

Quan,

Ou,

Long

et al. 2024

Analytica Chimica Acta

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A self-catalyzing strategy for co-immobilization of two distinct proteins at equimolar ratio: A case study of 3A and 2C to develop a chromatographic method for finding prospective dual-target compoundsfrom complex matrices

Quan,

Ou,

Long

et al. 2024

Analytica Chimica Acta

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Sequence-Directed Covalent Protein-RNA Linkages in a Single Step Using Engineered HUH-Tags

Smiley,

Babilonia-Díaz,

Krueger

et al. 2024

Preprint

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Replication-initiating HUH-endonucleases (Reps) are enzymes that form covalent bonds with single-stranded DNA (ssDNA) in a sequence specific manner to initiate rolling circle replication. These nucleases have been co-opted for use in biotechnology as sequence specific protein-ssDNA bioconjugation fusion partners dubbed ‘HUH-tags’. Here, we describe the engineering andin vitrocharacterization of a series of laboratory evolved HUH-tags capable of forming robust sequence-directed covalent bonds with unmodified RNA substrates. We show that promiscuous Rep-RNA interaction can be enhanced through directed evolution from nearly undetectable levels in wildtype enzymes to robust reactivity in final engineered iterations. Taken together, these engineered HUH-tags represent a promising platform for enabling site-specific protein-RNA covalent bioconjugation in vitro, potentially mediating a host of new applications and offering a valuable addition to the HUH-tag repertoire.

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HUH Endonuclease: A Sequence-specific Fusion Protein Tag for Precise DNA-Protein Conjugation

Cited by 2 publications

References 100 publications

A self-catalyzing strategy for co-immobilization of two distinct proteins at equimolar ratio: A case study of 3A and 2C to develop a chromatographic method for finding prospective dual-target compoundsfrom complex matrices

A self-catalyzing strategy for co-immobilization of two distinct proteins at equimolar ratio: A case study of 3A and 2C to develop a chromatographic method for finding prospective dual-target compoundsfrom complex matrices

Sequence-Directed Covalent Protein-RNA Linkages in a Single Step Using Engineered HUH-Tags

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