Multiplexed Nucleic Acid Programmable Protein Arrays
Rationale: Cell-free protein microarrays display naturally-folded proteins based on just-in-time in situ synthesis, and have made important contributions to basic and translational research. However, the risk of spot-to-spot cross-talk from protein diffusion during expression has limited the feature density of these arrays.
Methods: In this work, we developed the Multiplexed Nucleic Acid Programmable Protein Array (M-NAPPA), which significantly increases the number of displayed proteins by multiplexing as many as five different gene plasmids within a printed spot.
Results: Even when proteins of different sizes were displayed within the same feature, they were readily detected using protein-specific antibodies. Protein-protein interactions and serological antibody assays using human viral proteome microarrays demonstrated that comparable hits were detected by M-NAPPA and non-multiplexed NAPPA arrays. An ultra-high density proteome microarray displaying > 16k proteins on a single microscope slide was produced by combining M-NAPPA with a photolithography-based silicon nano-well platform. Finally, four new tuberculosis-related antigens in guinea pigs vaccinated with Bacillus Calmette-Guerin (BCG) were identified with M-NAPPA and validated with ELISA.
Conclusion: All data demonstrate that multiplexing features on a protein microarray offer a cost-effective fabrication approach and have the potential to facilitate high throughput translational research.
- Author (aut): Yu, Xiaobo
- Author (aut): Song, Lusheng
- Author (aut): Petritis, Brianne
- Author (aut): Bian, Xiaofang
- Author (aut): Wang, Haoyu
- Author (aut): Viloria, Jennifer
- Author (aut): Park, Jin
- Author (aut): Bui, Hoang
- Author (aut): Li, Han
- Author (aut): Wang, Jie
- Author (aut): Liu, Lei
- Author (aut): Yang, Liuhui
- Author (aut): Duan, Hu
- Author (aut): McMurray, David N.
- Author (aut): Achkar, Jacqueline M.
- Author (aut): Magee, Mitch
- Author (aut): Qiu, Ji
- Author (aut): LaBaer, Joshua
- Contributor (ctb): Biodesign Institute