Multiplexed Nucleic Acid Programmable Protein Arrays

Yu, Xiaobo; Song, Lusheng; Petritis, Brianne; Bian, Xiaofang; Wang, Haoyu; Viloria, Jennifer; Park, Jin; Bui, Hoang; Li, Han; Wang, Jie; Liu, Lei; Yang, Liuhui; Duan, Hu; McMurray, David N.; Achkar, Jacqueline M.; Magee, Mitch; Qiu, Ji; LaBaer, Joshua

To determine whether NAPPA and M-NAPPA detect similar protein-protein interactions, both arrays were programmed to display proteins that are known to interact with the tumor suppressor protein Rb1. The arrays were then probed with a Rb1 query protein fused to HaloTag, and interactions were detected using an anti-HaloTag antibody. The Rb1-HaloTag query protein bound to several targets (red arrow) on NAPPA and M-NAPPA arrays; the query also bound to diffused targets outside of each spot, which appear as a "ring" around each feature (Figure 4A) . In Figure 4B , we used a flower pattern diagram to depict the multiplexed proteins on M-NAPPA (large central circle) and the deconvoluted individual proteins on NAPPA (five small connecting circles) (Materials and Methods). The blue gradient within the spot indicates target binding to the Rb1 query protein, whereas reactivity to the "ring" [20, 33] is indicated by a red circle around the spot. Using custom defined criteria, where the target-to-"non-spot" signal ratio is > 2 and the ring score is > 3, we found that 5 and 6 hits were identified on NAPPA and M-NAPPA, respectively, out of the 30 possible candidate target proteins ( Table S2, Table S3 ). Five of the 6 hits on M-NAPPA were E1A, HPV11-E7, HPV16-E7, HPV18-E7 and HPV33-E7, which agrees with previous studies [38] [39] [40] . The sixth hit on M-NAPPA was not detected with NAPPA, thus suggesting that the hit may be a false positive. To further examine the utility of M-NAPPA to test protein-protein interactions, additional interactions were analyzed with 35 displayed proteins on NAPPA and M-NAPPA using HaloTagged-Jun, -Fos, and -LidA queries. Jun, Fos and LidA bound to their expected interaction partners (i.e., Fos, Jun and three Rab family proteins, respectively) on both M-NAPPA and NAPPA arrays (Figure S9) . Regarding the protein interactions that were identified, the spot-to-spot and zone-to-to zone CVs were 5.65±2.69% and 5.75±3.86% for NAPPA, respectively, and 2.55±2.56% and 3.11±3.46% for M-NAPPA, respectively (Table S5) .