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

M-NAPPA would be useful in unbiased HT screening studies, such as protein-protein interactions, protein-DNA interactions, discovery of drug binding target as well as (auto)antibody biomarkers for a variety of human diseases. However, it should be noted that there are situations in which using a non-multiplexed array format would be more appropriate. For example, NAPPA should be used when investigating protein functions or when the number of proteins to be screened is low. Additional attributes of M-NAPPA should be considered as well. Large, multiplexed proteins (> 65 kDa) on M-NAPPA are displayed at a lower level (37 -40%) than their non-multiplexed counterparts ( Figure 2C ). This issue could be resolved by increasing plasmid DNA concentration before printing or reducing multiplicity per spot. Alternatively, since large proteins represent a small fraction of the proteome, a hybrid array containing multiplexed spots with plasmids encoding for proteins with low to moderate MWs and non-multiplexed spots for large proteins (> 65 kDa) could be employed. In addition, PTMs that occur during cell-free protein expression may affect the protein display or activity on M-NAPPA arrays [42] . We have observed that the human expression system contains the ability to phosphorylate some proteins (data not shown); other types of PTMs (e.g., glycosylation, acetylation) by the expression system are not well known or reported. In our studies, PTMs did not appear to affect protein expression, protein-protein interactions, or the identification of serological antigens on M-NAPPA when compared to NAPPA (Figure 2, Figures 4-7 and Figure S7 ).