HistoPaints: Antibody Surrogates Applicable to Solid Tissues

Comprehensive identification of each distinct cell type in developing and adult tissues, their distinct signaling capabilities, developmental potential and physiology, and their spatial and lineage relationships is a central challenge in modern vertebrate biology. Much of our current understanding of the adaptive immune system derives from the use fluorescence activated cell sorting (FACS) to systematically identify, and then characterize, the diverse differentiated cell types that comprise this system. In principle, the same strategy could be applied to cataloging the molecular diversity, demographics and spatial organization of individual cells in solid tissues, by using in situ microscopic imaging for multidimensional molecular classification and characterization of these cells as FACS sorting techniques were historically used to hierarchically classify blood-born cells. A collection of specialized high-quality affinity reagents is a critical requirement. This ancillary proposal aims to test a general strategy for quickly and inexpensively obtaining small-molecule affinity reagents that mark specific cellular targets. Small molecules are highly desirable affinity reagents because: (a) they overcome diffusion barriers that limit or slow antibody access, (b) they can be engineered to bear modular chemical handles for easy tethering to fluorophores and straightforward affinity purification of cells, (c) their specificity and binding affinity can be radically increased by linking compounds with complementary binding properties, (d) they are easily scalable, so that a lifetime supply can be synthesized cheaply, stored on a shelf at room temperature, and distributed to many labs, and (e) they have potential utility for marking cytoplasmic features in living cells. The cost of high-throughput screening/medicinal chemistry prohibits its use for the development of new histological stains or imaging agents. We will instead apply a new DNA-programmed combinatorial chemistry/in vitro small-molecule evolution technology [1] to the problem of obtaining imaging agents for a collection of key protein regulators of lung development. We hope to identify cells and other features in solid lung tissue with at least the specificity achieved by FACS. This work tests a new experimental paradigm for proteome-wide molecular detection, and the findings will generalize to other solid organs, and to other areas of basic science.

Copyright ©2013 NHLBI Progenitor Cell Biology Consortium.

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