ArticleNucleic acids research2006
In vitro evolution of single-chain antibodies using mRNA display.
Article in Nucleic acids research, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 33 papers.
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Who cites it
33 citing papers in PubMed, 86 citations in OpenAlex.
- Approaches for Studying Context Specificity of Translation Inhibitor Action.International journal of molecular sciences · 2026Review
- Encoding and display technologies for combinatorial libraries in drug discovery: The coming of age from biology to therapy.Acta pharmaceutica Sinica. B · 2024Review
- mRNA Display Pipeline for Protein Biosensor Construction.ACS sensors · 2024Article
- Construction and Validation of Chicken Immune scFv Antibody Library againstMicroorganisms · 2024Article
- Hepatitis B virus evades the immune system by suppressing the NF-κB signaling pathway with DENND2A.Microbiology spectrum · 2024Article
- Article
- Cell-Free Display Techniques for Protein Evolution.Advances in biochemical engineering/biotechnology · 2023Article
- Directing evolution of novel ligands by mRNA display.Chemical Society reviews · 2021Review
- Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation.International journal of molecular sciences · 2019Review
- Nucleic Acid-Barcoding Technologies: Converting DNA Sequencing into a Broad-Spectrum Molecular Counter.Angewandte Chemie (International ed. in English) · 2019Review
- High-throughput screening of biomolecules using cell-free gene expression systems.Synthetic biology (Oxford, England) · 2018Review
- Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption.PloS one · 2016Article
- Endosomal escape efficiency of fusogenic B18 and B55 peptides fused with anti-EGFR single chain Fv as estimated by nuclear translocation.Journal of biochemistry · 2016Article
- Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step.International journal of molecular sciences · 2015Article
- A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.PloS one · 2015Article
- In vitro Fab display: a cell-free system for IgG discovery.Protein engineering, design & selection : PEDS · 2014Article
- In vitro selection of proteins with desired characteristics using mRNA-display.Methods (San Diego, Calif.) · 2013Article
- Streamlined protocol for mRNA display.ACS combinatorial science · 2013Article
- Attainment of 15-fold higher affinity of a Fusarium-specific single-chain antibody by directed molecular evolution coupled to phage display.Molecular biotechnology · 2012Article
- Generation of a highly reactive chicken-derived single-chain variable fragment against Fusarium verticillioides by phage display.International journal of molecular sciences · 2012Article
Corrections and comments
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Here we describe the application of the in vitro virus mRNA display method, which involves covalent linkage of an in vitro-synthesized antibody (phenotype) to its encoding mRNA (genotype) through puromycin, for in vitro evolution of single-chain Fv (scFv) antibody fragments. To establish the validity of this approach to directed antibody evolution, we used random mutagenesis by error-prone DNA shuffling and off-rate selection to improve the affinity of an anti-fluorescein scFv as a model system. After four rounds of selection of the library of mRNA-displayed scFv mutants, we obtained six different sequences encoding affinity-matured mutants with five consensus mutations. Kinetic analysis of the mutant scFvs revealed that the off-rates have been decreased by more than one order of magnitude and the dissociation constants were improved approximately 30-fold. The antigen-specificity was not improved by affinity maturation, but remained similar to that of the wild type. Although the five consensus mutations of the high-affinity mutants were scattered over the scFv sequence, analysis by site-directed mutagenesis demonstrated that the critical mutations for improving affinity were the two that lay within the complementarity determining regions (CDRs). Thus, mRNA display is expected to be useful for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.