With the growing interest in the field of proteomics, there is a need to generate more protein affinity reagents than is possible using standard antibody approaches, which tend to be costly and time-consuming due in large measure to the need for animal immunization. While alternative binders based on engineered immunoglobulin domains, protein scaffolds, and aptamers can be easier to obtain, they still require multiple rounds of time- and labor-intensive in vitro selection and amplification, steps that are not amenable to high-throughput methodologies.
In addition, because alternative binding reagents based on small molecule ligands are commonly selected from combinatorial libraries, they typically have only moderate binding affinities for a target protein when compared to antibodies. Increased binding affinity can be achieved by attaching two or more ligands to a tethering molecule to create a higher affinity, multivalent binding reagent.
In a recent issue of the Journal of the American Chemical Society, Williams et al. describe a novel strategy for the scalable production of such synthetic antibodies—which they term synbodies—by tethering two peptide ligands into a single high-affinity bivalent protein binding reagent using DNA.
The authors first used peptide microarrays to isolate moderate affinity peptide binders to a specific target protein. Two peptides that bind to different sites on the target protein were then tethered to a short DNA oligonucleotide by amine coupling to modified bases at various positions, with each peptide on a separate strand. The optimal distance and orientation of the two peptides for generating the strongest binding synbody was determined using surface plasmon resonance (SPR) analysis where all possible pairs (both hetero- and homo-) of peptides attached to the DNA at a variety of locations were screened on a chip.
Using the yeast Gal80 regulatory protein and human transferrin as targets, pairs of peptides with individual binding affinities in the low micro-molar range were transformed into synbodies with ~1000-fold higher binding affinities, levels of affinity comparable to monoclonal antibodies. These synbodies were shown to work in ELISA and protein pull-down assays, further demonstrating their effectiveness as substitutes for antibodies.
Williams et al. Creating protein affinity reagents by combining peptide ligands on synthetic DNA scaffolds. J. Am Chem Soc. 2009 Nov 6. [Epub ahead of print; DOI: 10.1021/ja9051735]