Sortase enzymes are cysteine transpeptidases that anchor surface proteins to the cell wall of Gram-positive bacteria. These enzymes are widely used in protein engineering applications because they…

Sortases are cysteine transpeptidases located on the surface of Gram-positive bacteria. These critic

Sortases are cysteine transpeptidases located on the surface of Gram-positive bacteria. These critic

Wild-type sortase A is an important virulence factor displaying a diverse array of proteins on the surface of bacteria and is engineered to allow protein ligation. Here, authors discover a previously unknown interaction between multiple classes of wild-type sortases and engineered sortase A and oligonucleotides. This property enables sortase-mediated oligonucleotide mammalian cell labeling, which can be applied in multiplexing samples for single-cell sequencing. Abstract Wild-type sortase A is an important virulence factor displaying a diverse array of proteins on the surface of bacteria. This protein display relies on the transpeptidase activity of sortase A, which is widely engineered to allow protein ligation and protein engineering based on the interaction between sortase A and peptides. Here an unknown interaction is found between sortase A from Staphylococcus aureus and nucleic acids, in which exogenously expressed engineered sortase A binds oligonucleotides in vitro and is independent of its canonical transpeptidase activity. When incubated with mammalian cells, engineered sortase A further mediates oligonucleotide labeling to the cell surface, where sortase A attaches itself and is part of the labeled moiety. The labeling reaction can also be mediated by many classes of wild-type sortases as well. Cell surface GAG appears involved in sortase-mediated oligonucleotide cell labeling, as demonstrated by CRISPR screening. This interaction property is utilized to develop a technique called CellID to facilitate sample multiplexing for scRNA-seq and shows the potential of using sortases to label cells with diverse oligonucleotides. Together, the binding between sortase A and nucleic acids opens a new avenue to understanding the virulence of wild-type sortases and exploring the application of sortases in biotechnology.