Abstract
We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.
Highlights
The pace of improvement in DNA sequencing technologies is far outpacing Moore’s Law [1]
Many new whole genome sequences are rapidly becoming available for analysis. This has created a pressing need for new technologies that enable the translation of genomic sequence information into information about protein function at the level of the proteome. We address this need by reporting a new approach for producing and assaying large, customizable sets of peptide-cDNA conjugates that combines efficient enzymatic synthesis of in silico designed peptide sequences with intrinsically parallel assay formats designed for readout by DNA sequencing
A random combinatorial library of 10-mer peptides would comprise 20‘10, or approximately 10‘13, sequences. This is larger than the coding content of the,3610‘9 base pair human genome by several orders of magnitude. We address this limitation by making use of microarray-based design and synthesis of custom DNA oligonucleotide sequences to encode specific peptide sequences of interest
Summary
The pace of improvement in DNA sequencing technologies is far outpacing Moore’s Law [1]. A variety of molecular biology techniques for the production and screening of large numbers of proteins or peptides linked to their encoding nucleic acid sequences has been developed, primarily to enable the discovery of high affinity binders to targets of interest These methods, which include phage display [2,3,4,5,6,7], PROfusionTM technology [8,9], and others [10,11,12,13,14,15,16,17,18], provide means to identify enriched proteins via a nucleic acid tag and avoid the limitations of chemical synthesis (e.g. racemization, limited peptide length, complex multistep synthesis, and use of toxic chemicals) [19]
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