Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies

Kondo, Tohru; Eguchi, Minori; Tsuzuki, N.; Murata, Naoya; Fujino, Tomoshige; Hayashi, Gosuke; Murakami, Hiroshi

doi:10.21769/bioprotoc.4125

Cited by 2 publications

(2 citation statements)

References 24 publications

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“…The resulting complex was used directly in the first-round of selection. The C6-AM2 and C12-AM2 DNA library was prepared using a similar procedure of previously published articles (Kondo et al 2020(Kondo et al , 2021b. To prepare A-fragment DNA from the monobody library, FN3F-QAN.F1 (1 μM), FN3F.F3short39 (1 μM), and Mono-bodyHL1Co10 (0.5 μM) or MonobodyHL1Co12 (0.5 μM) were ligated by T4 DNA ligase (150 μl in total, 150 pmol for each oligonucleotide) with the assistance of MonoE1c1NH2-NNN.R25 (2 μM), and MonoE1c2-NNN1-NH2.24 (2 μM).…”

Section: Preparation Of Monobody Mrna Librariesmentioning

confidence: 99%

Monobodies with potent neutralizing activity against SARS-CoV-2 Delta and other variants of concern

et al. 2022

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Neutralizing antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are useful for patients’ treatment of the coronavirus disease 2019 (COVID-19). We report here affinity maturation of monobodies against the SARS-CoV-2 spike protein and their neutralizing activity against SARS-CoV-2 B.1.1 (Pango v.3.1.14) as well as four variants of concern. We selected matured monobodies from libraries with multi-site saturation mutagenesis on the recognition loops through in vitro selection. One clone, the C4-AM2 monobody, showed extremely high affinity (KD < 0.01 nM) against the receptor-binding domain of the SARS-CoV-2 B.1.1, even in monomer form. Furthermore, the C4-AM2 monobody efficiently neutralized the SARS-CoV-2 B.1.1 (IC50 = 46 pM, 0.62 ng/ml), and the Alpha (IC50 = 77 pM, 1.0 ng/ml), Beta (IC50 = 0.54 nM, 7.2 ng/ml), Gamma (IC50 = 0.55 nM, 7.4 ng/ml), and Delta (IC50 = 0.59 nM, 8.0 ng/ml) variants. The obtained monobodies would be useful as neutralizing proteins against current and potentially hazardous future SARS-CoV-2 variants.

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Section: Preparation Of Monobody Mrna Librariesmentioning

confidence: 99%

Monobodies with potent neutralizing activity against SARS-CoV-2 Delta and other variants of concern

et al. 2022

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“…For example, Kondo, et al . recently reported the synthesis of a monobody library with 10 13 variants that required multiple 15 mL PCR reactions followed by digestion, ligation, and a final amplification in a 60 mL PCR reaction [ 13 , 14 ]. According to the authors, the entire process took a whopping six months to complete [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

A PCR-free rapid protocol for one-pot construction of highly diverse genetic libraries

Woolley

Chen²

2022

PLoS ONE

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In vitro protein display methods can access extensive libraries (e.g., 1012–1014) and play an increasingly important role in protein engineering. However, the preparation of large libraries remains a laborious and time-consuming process. Here we report an efficient one-pot ligation & elongation (L&E) method for sizeable synthetic library preparation free of PCR amplification or any purification steps. As a proof of concept, we constructed an ankyrin repeat protein templated synthetic library with 1011 variants in 150 μL volume. The entire process from the oligos to DNA template ready for transcription is linearly scalable and took merely 90 minutes. We believe this L&E method can significantly simplify the preparation of synthetic libraries and accelerate in vitro protein display experiments.

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Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies

Cited by 2 publications

References 24 publications

Monobodies with potent neutralizing activity against SARS-CoV-2 Delta and other variants of concern

Monobodies with potent neutralizing activity against SARS-CoV-2 Delta and other variants of concern

A PCR-free rapid protocol for one-pot construction of highly diverse genetic libraries

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