cDNA TRAP display for rapid and stable <i>in vitro</i> selection of antibody-like proteins

Kondo, Tohru; Eguchi, Minori; Kito, Seita; Fujino, Tomoshige; Hayashi, Gosuke; Murakami, Hiroshi

doi:10.1039/d0cc07541h

Cited by 9 publications

(10 citation statements)

References 33 publications

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“…The first and second bases of codons were limited to V to avoid hydrophobic residues for further application as linkers to connect protein or peptide libraries to mRNAs. To avoid the undesired effect of amino acid sequences coded by VVN codons on pull-down efficiency, biotin was incorporated at the N -terminus of fibronectin using the Flexizyme system 36-38 , and only the fibronectin-PuL/mRNA complexes were isolated by the biotin pull-down 26,39 . An enrichment factor was calculated as described above.…”

Section: Resultsmentioning

confidence: 99%

Comprehensive analysis of the effect of mRNA sequences on translation efficiency and accuracy

Umemoto

Kondo

Fujino

et al. 2022

Preprint

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Messenger ribonucleic acid (mRNA) sequences influence the translation efficiency and accuracy. To increase our knowledge of how mRNA sequences affect ribosome translation and apply the obtained information to improve the mRNA display method, we conducted a comprehensive analysis of the effect of mRNA sequences on the translation. Translation efficiency depended strongly on the three codons following the start codon. Furthermore, the codons at the ribosomal E- and P-sites strongly influence the misreading of the A-site blank codon by near-cognate transfer RNA. The purine base after the blank codon also induced a higher misread rate than that with a pyrimidine base. Based on these findings, we demonstrated construction of highly diverse monobody and macrocyclic peptide libraries that would be useful in developing functional peptides and proteins in the future.

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Section: Resultsmentioning

confidence: 99%

Comprehensive analysis of the effect of mRNA sequences on translation efficiency and accuracy

Umemoto

Kondo

Fujino

et al. 2022

Preprint

Self Cite

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“…Finally, annealing alone gives a sufficiently stable connection if the complementary sequence is long enough and this allows a much faster protocol (termed ‘TRAP display’ by the authors) [18] . Furthermore, using a branched linker in this context again allows it to serve as reverse transcription primer (termed ‘cDNA‐TRAP display’ by the authors) [19] . These various techniques will collectively be referred to as simply ‘mRNA display’ in this review, but in cases where one approach requires special consideration it will be discussed with the name above.…”

Section: Variants Of Mrna Display and Dna‐encoded Librariesmentioning

confidence: 99%

Opportunities for Expanding Encoded Chemical Diversification and Improving Hit Enrichment in mRNA‐Displayed Peptide Libraries

2022

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DNA‐encoded small‐molecule libraries and mRNA displayed peptide libraries both use numerically large pools of oligonucleotide‐tagged molecules to identify potential hits for protein targets. They differ dramatically, however, in the ‘drug‐likeness’ of the molecules that each can be used to discover. We give here an overview of the two techniques, comparing some advantages and disadvantages of each, and suggest areas where particularly mRNA display can benefit from adopting advances developed with DNA‐encoded small molecule libraries. We outline cases where chemical modification of the peptide library has already been used in mRNA display, and survey opportunities to expand this using examples from DNA‐encoded small molecule libraries. We also propose potential opportunities for encoding such reactions within the mRNA/cDNA tag of an mRNA‐displayed peptide library to allow a more diversity‐oriented approach to library modification. Finally, we outline alternate approaches for enriching target‐binding hits from a pooled and tagged library, and close by detailing several examples of how an adjusted mRNA‐display based approach could be used to discover new ‘drug‐like’ modified small peptides.

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“…Non-immunoglobulin–based protein derived from the 10th type III domain of human fibronectin, monobody or adnectin, is one of the smallest back-bone proteins used for in vitro selection and is well expressed in a bacterial system ( Koide et al 1998 , 2012 ; Wojcik et al, 2010 ; Lipovsek 2011 ). In a previous publication, by using the transcription-translation coupled with puromycin-linker (TRAP) display ( Ishizawa et al, 2013 ; Kondo et al, 2020 , 2021a ), we developed three monobodies (C4, C6b, and C12b), which simultaneously bind to a distinct epitope in the RBD with nM or sub-nM affinity ( Kondo et al, 2020 ). The C4 and C6b monobodies inhibited the ACE2 and RBD interaction and, in addition, the C6b monobody showed moderate neutralizing activity against the SARS-CoV-2 B.1.1 (Pango v.3.1.14, referred to as the “wild type” in this study) ( IC 50 = 0.5 nM).…”

Section: Introductionmentioning

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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cDNA TRAP display for rapid and stable in vitro selection of antibody-like proteins

Abstract: We developed a cDNA TRAP display for the rapid selection of antibody-like proteins in various conditions. By modifying the original puromycin linker in the TRAP display, a monobody was covalently...

Cited by 9 publications

References 33 publications

Comprehensive analysis of the effect of mRNA sequences on translation efficiency and accuracy

Comprehensive analysis of the effect of mRNA sequences on translation efficiency and accuracy

Opportunities for Expanding Encoded Chemical Diversification and Improving Hit Enrichment in mRNA‐Displayed Peptide Libraries

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

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