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

Since the multiplex concept to increase feature density was successful in detecting protein-protein interactions and serological antibody responses on planar microarrays, we wanted to determine whether M-NAPPA could also be applied to a nano-well microarray platform. We previously increased feature number by printing plasmids into photolithography-etched silicon nano-wells to create a high-density NAPPA (HD-NAPPA) platform [41] . HD-NAPPA can have as many as 10k features per slide, and has successfully detected antiviral antibodies in autoimmune diseases with 761 different proteins displayed on the array in quadruplicate. These tiny wells hold only 1200 pL and use only 0.12 ng of plasmid DNA. We applied the multiplex concept to HD-NAPPA using a mixture of plasmids encoding for IA-2, GAD2 and p53 proteins. We then detected their expression and display using specific antibodies; all of these proteins were readily detectable when printed as a three-plexed mixture ( Figure S10) . We then multiplexed 4,045 tuberculosis (TB) ORFs [32] onto HD-NAPPA microarrays as four separate subarrays using three gene plasmids per well (M=3), resulting in an M-HD-NAPPA microarray displaying > 16k proteins on a single slide. This lower multiplicity was based on the mathematical model ( Figure 1C ) that took into account that the high number of conserved proteins in endemic, non-pathogenic mycobacterial species results in a higher hit rate (~10% [37] ). Over 95% of the spots generated a signal that was at least 10 SDs above the background, which indicates that the vast majority of proteins were well-expressed and displayed (Figure 6A) , with a correlation of R = 0.90 across technical replicates (Figure 6A-C) . Antibody reactivity from TB patient sera was observed with M-HD-NAPPA (Figure 6D) . The technical reproducibility of these immune-dominant antigens across different M-NAPPA arrays using the same sera was very high, with a correlation of R = 0.98 (Figure 6D-F) . All immune-dominant antigens identified with M-HD-NAPPA screening were then deconvoluted in the verification step using single protein NAPPA ( Figure 6G ) and validated with RAPID-ELISA as previously described [19] (Figure 6H ). We screened the sera from guinea pigs immunized with Bacillus Calmette-Guerin (BCG), a TB vaccine, using M-HD-NAPPA TB proteome microarrays. The aim of this experiment was to identify potential protective antibodies induced with BCG. The representative fluorescence images are shown in Figure 7A . Compared to the control mock sera pool using PBS buffer (n=5), four features on M-NAPPA arrays showed increased signals with the BCG samples (n=4) (Figure 7B) . To deconvolute and validate those targets, we repeated the serological assay for those candidate proteins, along with two non-responsive control proteins (Rv2077A and Rv2682c), using RAPID-ELISA and the individual sera from the guinea pigs. The antibody levels of four antigens (Rv3405c, Rv1078, Rv2853 and Rv0928) in BCG-vaccinated guinea pigs were significantly higher than that of the PBS control with a p-value <0.01 ( Figure 7C, Figure S11 ). According to the Tuberculist database (http://tuberculist.epfl.ch/), these proteins are involved in regulation, cell wall and cell processes, and are considered to be in the proline-glutamic acid / proline-protein-glutamic acid (PE/PPE) protein families ( Figure 7D ). A primary advantage of cell-free protein microarrays is that the arrays have a long shelf life. We compared the prot