In recent years new nucleic acid and protein-based combinatorial molecules have attracted the attention of researchers working in various areas of science, ranging from medicine to analytical chemistry. These molecules, called aptamers, have been proposed as alternatives to antibodies in many different applications. The aim of this Review is to illustrate the peculiarities of these combinatorial molecules which have initially been explored for their importance in molecular medicine, but have enormous potential in other biotechnological fields historically dominated by antibodies, such as bioassays. A description of these molecules is given, and the methods for their selection and production are also summarized. Moreover, critical aspects related to these molecules are discussed.

Nucleic acid and peptide aptamers: fundamentals and bioanalytical aspects.

Peptide aptamers as guides for small-molecule drug discovery

A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using FRET between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types.

https://www.pnas.org/content/pnas/103/49/18458.full.pdf

Intracellular protein interaction mapping with FRET hybrids.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2802%2902928-9

Identification of the first Rho–GEF inhibitor, TRIPα, which targets the RhoA-specific GEF domain of Trio

Peptide aptamers are combinatorial proteins that specifically bind intracellular proteins and modulate their function. They are powerful tools to study protein function within complex regulatory networks and to guide small-molecule drug discovery. Here we describe methodological improvements that enhance the yeast two-hybrid selection and characterization of large collections of peptide aptamers. We provide a detailed protocol to perform high-efficiency transformation of peptide aptamer libraries, in-depth validation experiments of the bait proteins, high-efficiency mating to screen large numbers of peptide aptamers and streamlined confirmation of the positive clones. We also describe yeast two-hybrid mating assays, which can be used to determine the specificity of the selected aptamers, map their binding sites on target proteins and provide structural insights on their target-binding surface. Overall, 12 weeks are required to perform the protocols. The improvements on the yeast two-hybrid method can be also usefully applied to the screening of cDNA libraries to identify protein interactions.

Selection and characterization of large collections of peptide aptamers through optimized yeast two-hybrid procedures

Publisher Summary A new class of dominant agents has been developed to facilitate the analysis of processes in diploid and genetically intractable organisms. These molecules are termed “peptide aptamers” because of their similarity to nucleic acid aptamers. Peptide aptamers are defined as antibody-like recognition agents that consist of conformationally constrained peptides displayed on the surface of scaffold proteins and that are distinguished from peptides of variable sequence displayed that are not constrained at both ends by the scaffold. Peptide aptamers are designed to inhibit cellular processes by interacting with proteins and disrupting their biological functions. Combinatorial libraries of peptide aptamers in principle contain aptamers that bind almost any protein target. Peptide aptamers specific for numerous proteins have been isolated by using the yeast two-hybrid system. The fact that peptide aptamers are typically selected in vivo by two-hybrid methods increases the likelihood that the molecules will function in vivo . In support of this notion, peptide aptamers function as reverse dominant agents in both mammalian cells and Drosophila melanogaster . This chapter describes how to obtain and characterize peptide aptamers for both the reverse and forward analysis of cellular processes.

Selection of genetic agents from random peptide aptamer expression libraries.

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Selection of genetic agents from random peptide aptamer expression libraries.