FIG 5.

Screening of compounds for inhibition of cleavage of the B19V Ori by NS1N using FAM-labeled B19V Ori20. (A) FAM-based in vitro nicking assay. Each of the 71 compounds was incubated with NS1N at 10 µM in the nicking buffer at 37°C for 16 to 18 h. The fluorescence intensity of each sample was detected on a microplate reader. Ori20 without NS1N was set up as the background, and Ori20 with NS1N and DMSO were set up as positive controls. We set up inhibition of 80% of the relative fluorescence intensity as a cutoff value for the inhibition of NS1N nicking. (B) Comparison of the 25 chosen compounds between the radioactive and fluorescent nicking assays. At a final concentration of 10 µM, 8 compounds showed inhibition of >80% in the NS1N nicking of the ³²P-labeled Ori30 (in red), and 8 compounds were positive for the NS1N nicking of the FAM-labeled Ori20 (in green). The correlation coefficient (r) between the radioactive and fluorescent nicking assays was calculated using SPSS software. (C) Scatter plot and trend line of the radioactive nicking assay with the fluorescent nicking assay. The scatter plot, trend line, and R² value for the two assays were calculated using SPSS software.