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Potential of Antibody Therapy for Respiratory Virus Infections
Published in Sunit K. Singh, Human Respiratory Viral Infections, 2014
Tze-Minn Mak, Ruisi Hazel Lin, Yee-Joo Tan
Most display technologies share four key steps: cloning of genotypic diversity, coupling of genotype with phenotype, selective pressure (bio-panning), and amplification. Some of the commonly used display technologies are covered in the subsections below, with screening methodologies for in vivo techniques (ribosome or mRNA display and phage display) broadly similar, and in vitro techniques (bacterial display, yeast display, and mammalian cell display) utilizing flow cytometry as a main screening tool (Figure 10.5).
High-Throughput Screening for Probe Development
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
Kimberly A. Kelly, Fred Reynolds, Kelly R. Kristof
Taking the HTS process of SELEX one step further, Jack Szostak has developed another method known as mRNA display (47). One of the major problems with phage display is that the bacteriophage are limiting because of the bacterial transformation requirement. With mRNA display, translation is done in vitro so that the peptide or protein can be physically linked to the nucleic acid, so genotype still equals phenotype, but the process of translation has not gone through a complex biological system. The generation of the library begins with the synthesis of mRNA oligonucleotides that terminate with puromyocin, a peptidyl acceptor and a translation-terminating antibiotic. The mRNA is translated in vitro with a commercially available kit until it comes to the puromyocin, where it enters at the A site and forms a covalent bond with the nascent peptide or protein. The conjugates isolated after mRNA display were found to bind with an affinity in the low nanomolar range, as opposed to the micromolar affinities found after phage display. For example, 20 different aptamers were found to bind to streptavidin, a commonly screened biomarker, with an affinity between 110 nM and 2.4 nM, as opposed to “strep-tag” peptide (SNWSHPQFEK) found via phage display that has a binding affinity of about 13 μΜ (48).
Strategies for targeting undruggable targets
Published in Expert Opinion on Drug Discovery, 2022
Gong Zhang, Juan Zhang, Yuting Gao, Yangfeng Li, Yizhou Li
However, two inevitable issues exist: one is incompatibility to toxic, insoluble, or misfolded proteins and the other is the limitation of library size (less than ~109 due to transformation or transfection efficiency). Accordingly, cell-free display systems including ribosome display and mRNA display have been developed (Table 3). These display systems harness the biological translation machinery but fully in vitro. Ribosome display requires a reconstituted transcription/translation system, allowing for nascent protein libraries to connect with their encoding mRNA in the protein-ribosome-mRNA complex upon translation (Figure 2a). Cycling from several rounds of transcription–translation–panning–washing–elution, ribosome display can accomplish enrichment of high-affinity binding members[52]. mRNA display library is constructed in vitro with puromycin attached at 3ʹ-end. Puromycin, structurally similar to the aminoacyl-tRNA, stalls the translational process to form a covalent linkage between the translation product and the encoding mRNA (Figure 2a). The selection round subsequently undergoes alternative peptide stabilization, reverse transcription, solid-phase selection, and PCR to amplify the enriched candidate. mRNA display is a monovalent library where each peptide is tagged by the corresponding coding mRNA and could accommodate modified unnatural amino acids to a certain extent[53]. In addition, owing to the cell-free property, ribosome display, and mRNA display technologies enable constructions of huge libraries with up to 1014 members (Table 3).