Creating protein affinity reagents on a proteome-wide scale is the next grand challenge in human medicine. High quality protein capture reagents are critical for the elucidation of protein function, molecular diagnostics and even therapeutics. With the acceleration in protein discovery, we will require affinity reagents to nearly all proteins. Yet the cumbersome use of antibodies produced in animals is expensive, time consuming, and often fails for many antigens. Alternative approaches like single chain antibodies and aptamers solve some of these problems, but still require many rounds of selection to get high quality reagents. We have developed a new and innovative concept for creating bivalent affinity reagents called "DNA synbodies." We combine two peptides with moderate affinity (Kd<1 pM) to a target protein into a single molecule that binds its target with the low nM affinity. Central to this concept is the use of synthetic DNA as a rigid scaffold that positions one peptide on the sense strand and the second peptide on the antisense strand. The use of DNA as a scaffold allows simple and reliable adjustment of the spacing and angle between the two peptides. Our preliminary data demonstrates that DNA synbodies are straightforward to produce and bind their targets with low nM affinity. Moreover, their sequences and positions are known precisely, so they are readily renewable, easy and inexpensive to produce and store, and they can even be E-mailed to another lab. DNA synbodies have a well-defined chemical structure that is easy to modify with labels or tags and are compatible with common functional assays. In this application we will develop the methodology necessary to create a complete high throughput pipeline to produce DNA synbodies to human proteins. The research plan consists of four phases: target production, affinity reagent production, affinity reagent characterization and validation, and affinity reagent distribution. For each part, we will optimize the technology and develop standard operating procedures amenable to large-scale production. We will also establish a database that contains all relevant information, including DNA synbody composition, binding affinities, kinetic parameters, specificity-all of which we will compare to standard commercial antibodies.

In this proposal we will develop the methodology to create a complete high throughput pipeline to produce bivalent synthetic antibodies to human proteins. These synthetic antibodies will allow the study of protein function and may lead to an understanding of how this contributes to disease. Moreover, they may be useful for medical diagnostics and therapeutics.