Examples described here clearly illustrate that engineering of synthetic molecular interaction interfaces using nonantibody scaffolds is a mature branch of protein engineering. The breadth of available scaffolds and their demonstrated effectiveness in producing highly functional interfaces suggest that establishing another molecular scaffold will be an exercise of diminishing returns, unless such a scaffold offers a clearly distinct advantage over the current ones. Although these scaffold systems have been developed primarily for overcoming limitations of natural antibodies, structural and biophysical analyses of binders produced from these systems have expanded the types of protein recognition motifs beyond those that are present in natural proteins and have provided fundamental insights into the molecular mechanisms underlying protein-ligand interactions.

The common goal of scaffold development has been to establish a single, highly versatile system that consistently produces high affinity and high specificity binders to all types of targets. However, this "one size fits all" goal should probably be reexamined. As described previously, the interface topography is a major determinant of protein-protein interaction interfaces. Therefore, scaffolds presenting different paratope shapes are expected to have different binding preferences. Consequently, the most successful strategy might be to use a few distinct and complementary scaffolds, which should increase the probability of successfully generating highperformance binders to diverse targets. It is interesting that recent studies in the synthetic antibody field show that this type of scaffold diversity is highly effective (Persson et al. 2006; Cobaugh et al. 2008). Given the diverse and complementary nature of highly functional scaffold systems that are already available, it is likely that we, as a community, will be able to achieve this goal of establishing a small number of complementary scaffolds sufficient for most applications in the near future.

Applications of synthetic binders are still in their infancy. Synthetic binders can be in many instances superior alternatives to natural antibodies as affinity reagents. Particularly, the invariant scaffolds facilitate the construction of highly efficient generation pipelines that can be easily scaled up. This is because, unlike natural antibodies that contain many variations in the scaffold outside the CDRs, a standardized manipulation of all members from a synthetic binder library can be easily established. However, the true value of synthetic binders as novel tools lies in their applications in areas that are deemed impossible or extremely difficult with natural and synthetic antibodies. These include intracellular applications, such as intrabod-ies and protein tracking in live cells, and implementation of higher-order functionalities such as sensors. In the future, it is highly likely that synthetic binders will prove to be an enabling factor in many unforeseen applications.

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