The Transpeptidase Sortase A Binds Nucleic Acids and Mediates Mammalian Cell Labeling

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.

The red arrow points to the product bands.The membranes were incubated with a mouse antibiotin primary antibody and a goat anti-mouse secondary antibody.

Figure S3 .
Figure S3.Concentration-dependent cell labeling.A) Flow cytometry quantification of the Jurkat cells labeled with FITC-modified DNA oligonucleotides.B) Summary plot of MFIs (mean fluorescence intensities) and percentages of Jurkat cells labeled with FITC-modified DNA oligonucleotides at different concentrations.Experiment was repeated 3 times with similar results.

Figure S4 .
Figure S4.The fluorescence signals of the positively labeled cells were detectable 120 hours post labeling.A) The FITC, TAMRA, Cy5-modified DNA oligonucleotide was used to label cells, and the flourescent signals were quantified within 24 hours but in smaller time intervals.B) Summary plot of the MFI (mean fluorescence intensity) and the percentage of positively labeled cells at 24 hours.S: mgSrtA; O: DNA oligonucleotide.Data are represented as mean

Figure S5 .
Figure S5.The signals of labeled TAMRA-modified (A and B) and biotin-modified (C and

Figure S6 .
Figure S6.The sequence screening for preferred nucleotides in mgSrtA-mediated cell labeling.A) Schematic flowchart B) The weblogo of the screening.

Figure S7 .
Figure S7.K562 cells labeled with dsDNA.A) Both double-stranded (ds) and single-stranded (ss) DNA were used in cell labeling mediated by mgSrtA.0.5 million K562 cells were incubated with 20 μM mgSrtA at 37°C for 5 min and incubated for another 10 min in the presence of 40 nM oligonucleotides.Equimolar quantities of dsDNA and ssDNA were used in this quantification, in which each dsDNA molecule carried double the amount of biotin modification compared to ssDNA.B) Summary plot of K562 cells labeled with biotinmodified dsDNA and ssDNA.MFI, mean fluorescence intensity.S: mgSrtA.Experiment was conducted in triplicates.*** p < 0.001, **** p < 0.0001 by unpaired 2-tailed t-test.

Figure S8 .
Figure S8.mgSrtA mediates Jurkat cell labeling with Cy5-modified RNA oligonucleotides.A) Flow cytometry quantification of Jurkat cells labeled with Cy5-modified RNA oligonucleotides.B) Summary plot of Jurkat cells labeled with Cy5-modified RNA oligonucleotides at different concentrations.MFI, mean fluorescence intensity.0.5 million Jurkat cells were incubated with 20 μM mgSrtA at 37°C for 5 min and incubated for another 10 min in the presence of oligonucleotides.S: mgSrtA.Experiment was conducted in triplicates.

Figure S9 .
Figure S9.mgSrtA mediates K562 cell labeling with a biotin-modified peptide nucleic acid (PNA).A) Flow cytometry quantification of the K562 cells labeled with biotin-modified PNA.B) Summary plot of K562 cells labeled with biotin-modified PNA.MFI, mean fluorescence intensity.Data are represented as mean ± SD of three replicates.*** p< 0.001 by unpaired 2tailed t-test.0.5 million K562 cells were incubated with 20 μM mgSrtA at 37°C for 5 min and incubated for another 10 min in the presence of 500 nM PNA.

Figure S10 .
Figure S10.Cell labeling can be applied to a variety of cell lines.We applied the oligonucleotides to cells of multiple types in the presence of mgSrtA.A) Flow cytometry quantification of cultured cells of twelve different cell lines.B) Summary plot of the percentage of positively labeled cultured cells.Experiment was repeated 3 times with similar results.C) Summary plot of the normalized MFI (mean fluorescence intensity) of the cultured cells.Data are represented as mean ± SD of three replicates.D) Flow cytometry quantifications of cultured primary cells of seven different tissues.E) Summary plot of the percentage of positively labeled primary cells.Experiment was conducted in triplicates.F) Summary plot of the normalized MFI of the primary cells.Data are represented as mean ± SD of three replicates.S: mgSrtA; O: DNA oligonucleotide.

Figure S11 .
Figure S11.The efficiency of mgSrtA-mediated cell labeling measured in different buffers.A) K562 cells were incubated with mgSrtA and FITC-modified oligonucleotides in different buffers.B) Summary plot of K562 cells labeled by mgSrtA in different buffers.S: mgSrtA; O: FITC-modified oligonucleotides.MFI, mean fluorescence intensity.Data are represented as mean ± SD of three replicates.P-values are calculated using one-way ANOVA, ns: not significant, ** p < 0.01, **** p <0 .0001.PBS served as control.

Figure S12 .
Figure S12.The efficiency of mgSrtA-mediated cell labeling measured at different pH values.A) K562 cells were incubated with mgSrtA and FITC-modified oligonucleotides at different pH values.B) Summary plot of K562 cells labeled by mgSrtA at different pH values.S: mgSrtA; O: FITC-modified oligonucleotides.MFI, mean fluorescence intensity.Data are represented as mean ± SD of three replicates.ns: not significant, *** p< 0.001 by unpaired 2tailed t-test.

Figure S13 .
Figure S13.The efficiency of mgSrtA-mediated cell labeling measured at different temperatures.A) K562 cells were incubated with mgSrtA and TAMRA-modified oligonucleotides at 4°C or RT or 37°C.B) Summary plot of K562 cells labeled by mgSrtA at different temperatures.MFI, mean fluorescence intensity.Data are represented as mean ± SD of three replicates.

Figure S14 .
Figure S14.Quenching the labeling reaction by EDTA.A) K562 cells were incubated with mgSrtA and FITC-modified oligonucleotides in the presence of EDTA.B) Summary plot of mgSrtA-mediated cell labeling terminated at different EDTA concentrations.MFI, mean fluorescence intensity.Data are represented as mean ± SD of three replicates.

Figure S15 .
Figure S15.List of CellIDs, labeled cell types, and originating species used in multiplexed scRNA-seq.

Figure S17 .
Figure S17.mgSrtA incubated with the oligonucleotides with increased length.(the 4G, 6G, 8G, 15G, and 20G oligonucleotides were biotinylated at the 5' end).The red arrow points to the product bands.The membrane was incubated with a mouse anti-biotin primary antibody and a goat anti-mouse secondary antibody.

Figure S18 .
Figure S18.mgSrtA was pretreated with 2% SDS before incubated with the 4G DNA oligonucleotide (the DNA oligonucleotide was modfied by 5' TAMRA and 3' biotin).The red arrow points to the product band.The membrane was incubated with a mouse anti-biotin primary antibody and a goat anti-mouse secondary antibody.

Figure S19 .
Figure S19.MST assays for the quantitation of the affinity of binding between mgSrtA and Cy5-modified oligonucleotides.Error bars represent the mean ± SEM based on four replicates.

Figure S21 .
Figure S21.K562 cells were incubated with FITC-modified AALPETG peptide and mgSrtA mutants carrying the mutations H120A, C184A, or R197A and mgSrtA-triple.A) The signals of the labeled peptide and anchored mgSrtA or its mutants on cells were detected by flow cytometry.B) Summary plot of the signal of labeled FITC-modified peptide on K562 cells.MFI, mean fluorescence intensity.Data are represented as mean ± SD of three replicates.Pvalues are calculated using one-way ANOVA, ns: not significant, **** p <0 .0001.

Figure S22 .
Figure S22.mgSrtA-mediated cell labeling conducted on K562 cells that were pretreated with various proteinases.A) K562 cells were incubated with various proteinases st 37°C for 5 min before labeled with FITC-modified oligonucleotides.B) The signal of labeled oligonucleotides on cells.S: mgSrtA; O: FITC-modified oligonucleotides.MFI, mean fluorescence intensity.Data are represented as mean ± SD of three replicates.P-values are calculated using one-way ANOVA, ns: not significant, **** p <0 .0001.PBS served as control.

Figure S25 .
Figure S25.mgSrtA-mediated cell labeling conducted on K562, Jurkat, and 293T cells that were pretreated with chondroitinases.A) The K562, Jurkat, and 293T cells were pretreated with chondroitinases before labeled with Cy5-modified oligonucleotides.B) The signal of the labeled oligonucleotides on K562, Jurkat, and 293T cells.MFI, mean fluorescence intensity.Data are represented as mean ± SD of three replicates.P-values are calculated using one-way ANOVA, ns: not significant, * p < 0.05, ** p <0 .01.Cells were pretreated with 0.2 U/mL or 1 U/mL Chondroitinase in HBSS at 37C for 1 h.

Figure S27 .
Figure S27.mgSrtA-mediated cell labeling conducted on K562, Jurkat, and 293T cells that were pretreated with hyaluronidases.A) The K562, Jurkat, and 293T cells were pretreated with hyaluronidases before labeled with Cy5-modified oligonucleotides.B) The signal of the labeled oligonucleotides on K562, Jurkat, and 293T cells.MFI, mean fluorescence intensity.Data are represented as mean ± SD of three replicates.P-values are calculated using one-way ANOVA, ns: not significant.Cells were pretreated with 5 U/mL or 25 U/mL Hyaluronidase in HBSS at 37C for 1 h.

Figure S29 .
Figure S29.The reaction between mgSrtA and heparin in vitro.A) 10 μM mgSrtA incubated with 300 ng/μL biotin-modified heparin at 37C for 30 min in the presence of 100 μM Cu 2+ .mgSrtA was pretreated in 2% SDS at 95 C for 10 min and returned to room temperature before incubated with heparin.The red arrow points to the product bands.B) mgSrtA

Figure S30 .
Figure S30.K562 cells were labeled with biotin-modified heparin mediated by mgSrtA, mgSrtA-L200F, and mgSrtA-triple.mgSrtA-triple represents the mgSrtA mutant with H120A, C184A, and R197A mutations.A) Flow cytometry quantification of the labeled K562.B) The signal of the labeled heparin on K562 ceslls.MFI, mean fluorescence intensity.Data are represented as mean ± SD of three replicates.0.5 million K562 cells were incubated with 20 μM mgSrtA at 37°C for 5 min and incubated for another 10 min in the presence of 3 ng/μL heparin.

Figure S31 .
Figure S31.mgSrtA mediates E.coli labeling with oligonucleotides.A) Flow cytometry quantification of the E.coli labeled with Cy5-modified oligonucleotides.B) The percentage of positively labeled E.coli.S: mgSrtA; O: oligonucleotide.Data are represented as mean ± SD of three replicates.*** p< 0.001 by unpaired 2-tailed t-test.5 millions E.coli were incubated

Figure S34 .
Figure S34.The resultant docking model.A) Reported crystal structure of wild-type sortase A and peptide.B) Docking simulation of the 4G DNA oligonucleotide and mgSrtA.

Figure S35 .
Figure S35.The top hits of CRISPR screening of genes that facilitate LPETG cell labeling.Genes are ranked (x-axis) by p value (y-axis).

Figure S36 .
Figure S36.The colocalization of the labeled LPETG peptide and anchored mgSrtA observed by confocal microscopy.A) Confocal images of K562 cells labeled with FITC-modified LPETG peptide and incubated with anti-his PE antibody.Blue, nucleus.Green, peptide.Red, mgSrtA.Yellow, the colocalization of mgSrtA and the oligonucleotide.The white arrow points to the colocalization sites.Scale bar, 20 μm.B) Fluorescence intensity profiles along the yellow line in the merged image.

Figure S37 .
Figure S37.K562 cells were labeled with FITC-modified LPETG peptide.A) The signals of the labeled oligonucleotide and anchored WTSrtA or its mutants on cells were detected by flow cytometry.B) Summary plot of K562 cells labeled with FITC-modified LPETG peptide mediated by WTSrtA and its mutants.Data are represented as mean ± SD of three replicates.