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Multiplexed Nucleic Acid Programmable Protein Arrays.


ABSTRACT: 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.

SUBMITTER: Yu X 

PROVIDER: S-EPMC5667425 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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<b>Rationale:</b> Cell-free protein microarrays display naturally-folded proteins based on just-in-time <i>in situ</i> 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. <b>Methods:</b> In this work, we developed the Multiplexed Nucleic Acid Programmable Protein Array (M-NAPPA), which significantly increases the number of displa  ...[more]

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