ABSTRACT
Bacterial thioredoxin A is among the most widely used scaffolds for in vivo selection. Thioredoxin-based peptide aptamers (PA) represent a new class of biologically active molecules that have the potential to bind a given target in solution, and extracellular and intracellular conditions to satisfy the demanding applications required in the biomedical, and bioanalytical fields. The choice of scaffold protein is critical to creating a combinatorial PA library. The ideal scaffold is a compact, rigid, monomeric, stable protein core capable of displaying variable target interaction surfaces in a manner analogous to the immunoglobulin complementarity-determining region. Comprehensive biophysical and biochemical characterization, confirmation of target recognition, and detailed atomic resolution of the binding interface are required. The original concept of constrained PA inserts is based on the variable loop-binding determinant present on immunoglobulins. The reduced conformation diversity lowers the entropic cost of binding when the target-bound PA adopts a single conformation and contributes to the tight binding affinities exhibited by doubly constrained PAs.
