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 decrease the cost of high throughput screening experiments, we hypothesized that the plasmid cDNA encoding for different proteins could be multiplexed (by combining M different plasmids) within each feature to create a high-density array, M-NAPPA ( Figure 1A) . This multiplexed array could be implemented during the initial functional screen, testing entire proteomes (P proteins) using only a fraction of the features (P/M). Multiplexed hits identified during the screening step could then be de-convoluted in the subsequent verification step using the standard, non-multiplexed NAPPA array where each feature displays only one protein (i.e., M=1). The objectives of the second step would be to identify which proteins were responsible for the positive multiplexed signal and to verify whether the hits were real. This approach exploits the high flexibility of cell-free microarrays, in which arrays can be customized by simply re-arraying individual plasmids encoding for the multiplexed features-of-interest.