Remarkable progress in single cell genomics rests on the polymerase chain reaction. There is no analogous tool to probe the cell-to-cell distribution of protein diversity and posttranslational modification (PTM) patterns at the few- or single-cell level. If protein-capture arrays can be synthesized on a molecular scale, then the arrays themselves become reagents suitable for incubation with the contents of even a single cell. Molecular arrays could be used in titrations, giving a potentially enormous dynamic detection range and facilitating quantification of the protein content and PTMs for each generation of the progeny of a single parent cell. Nanotechnology already provides the components for such a system, and we propose to integrate four new technologies to develop self-assembled nanoarrays for protein analysis. The first is construction of an array of single molecule probes arranged at nanometer density using nucleic acid self-assembly. The arrays are themselves giant molecules and are used like a reagent in a solution analysis, only being deposited onto a surface for a final readout. The second is a nanoscale readout using atomic force microscopy (AFM).The AFM is capable of reading out >30,000 array sites per minute. The third is the development of new molecular recognition elements based on aptamers and multivalent aptamers for proteins and their PTM variants. Because aptamers are synthetic and also nucleic acid sequences, individual aptamers can bind to a unique position on the array through nucleic acid hybridization and require no printing or lithography. The fourth is microfluidic technology to allow the nanoarrays to interact with lysed or intact cells, and to deliver the reacted arrays to predefined locations for readout. Our goal is to fill a unique niche in proteomics: parallel analysis of minute amounts of protein from small numbers of (potentially individual) cells. Can we make suitable ligands and will they work on arrays? Can we read the arrays accurately with AFM? Can we synthesize ligands that are highly selective for posttranslational modifications? What factors cause biodegradation of the arrays, and how can we control them while maintaining sensitivity and selectivity? What conditions are required to exploit the arrays for proteomics in a microfluidic system? What methodologies can we employ to deposit nanoliter solutions of reacted arrays at precise locations for AFM readout? These questions are the focus of this proposal. If successful, this work will make it possible to correlate nucleic acid diversity with the corresponding protein PTM diversity on a cell-by- cell basis, yielding, for the first time, data on the interplay between genome, gene expression, the environment and the spectrum of protein posttranslational modifications: the 'protein language'.
Probing the cell-to-cell distribution of protein diversity will provide critical information for understanding the development of living organisms and many diseases. Our goal is to develop a water soluble nanoarray system to open up to single-cell analysis the protein realm of the biochemical universe, uncovering new knowledge that will impact our understanding of how cells work and of how pathologies like cancer develop. This new technology will afford us a fuller view of the systems biology of the cell.
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