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.

SARS-CoV-2 proteome microarray for mapping COVID-19 antibody interactions at amino acid resolution

“…Serum screening using the SARS-CoV-2 proteome microarray can be performed in 1.5 hours while keeping a good dynamic range (∼2 orders) and sensitivity (94 pg/mL) (Figure 1b). This represents a significant decrease in time compared to the standard ∼ 18 hours using protein microarrays 17 The r correlation within an array and between different arrays were 0.9992 and 0.9978, respectively, demonstrating the high reproducibility the SARS-COV-2 proteome microarrays (Figure 1c).…”

Identification of Serum Biomarkers for Systemic Lupus Erythematosus Using a Library of Phage Displayed Random Peptides and Deep Sequencing

“…The use of phage displayed random peptide library allows the unbiased assessment of antibody binding to classify patients. Unlike protein microarrays (29,30), the display of random peptides is not restricted to proteins that have been cloned and can be expressed recombinantly. Moreover, for studies involving a large number of samples, the cost per sample of our strategy is roughly two orders of magnitude less than that of microarray-based alternatives (31).…”

Protein microarray technology: Assisting personalized medicine in oncology (Review)

“…Due to this improved technology, M-NAPPA, an ultra-high density proteome microarray could be done having >16,000 proteins per slide. This multiplexing has improved features, and is a new protein microarray for high-throughput translational research (37).…”