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Theranostics. 2017 Sep 20;7(16):4057-4070. doi: 10.7150/thno.20151. eCollection 2017.

Multiplexed Nucleic Acid Programmable Protein Arrays.

Author information

1
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (PHOENIX Center, Beijing), Beijing Institute of Radiation Medicine, Beijing, 102206, China.
2
The Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA.
3
Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843, USA.
4
Department of Medicine, Albert Einstein College of Medicine, NY 10461, USA; Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

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.

KEYWORDS:

Antibody; Biomarker.; Cell-free protein microarray; Protein-protein interaction; Proteomics

PMID:
29109798
PMCID:
PMC5667425
DOI:
10.7150/thno.20151
[Indexed for MEDLINE]
Free PMC Article

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