Site-directed conjugation of single-stranded DNA to affinity proteins: quantifying the importance of conjugation strategy

Affinity protein-oligonucleotide conjugates are increasingly being explored as diagnostic and therapeutic tools. Despite growing interest, these probes are typically constructed using outdated, non-selective chemistries, and little has been done to investigate how conjugation to oligonucleotides inf...

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Published inChemical science (Cambridge) Vol. 15; no. 23; pp. 8982 - 8992
Main Authors Rocha Tapia, Andres, Abgottspon, Fabrice, Nilvebrant, Johan, Nygren, Per-Åke, Duclos Ivetich, Sarah, Bello Hernandez, Andres Javier, Thanasi, Ioanna A, Szijj, Peter A, Sekkat, Ghali, Cuenot, François M, Chudasama, Vijay, Aceto, Nicola, deMello, Andrew J, Richards, Daniel A
Format Journal Article
LanguageEnglish
Published England Royal Society of Chemistry 12.06.2024
The Royal Society of Chemistry
Subjects
Affinity
Chemistry
Conjugates
Conjugation
Oligonucleotides
Proteins
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Summary:Affinity protein-oligonucleotide conjugates are increasingly being explored as diagnostic and therapeutic tools. Despite growing interest, these probes are typically constructed using outdated, non-selective chemistries, and little has been done to investigate how conjugation to oligonucleotides influences the function of affinity proteins. Herein, we report a novel site-selective conjugation method for furnishing affinity protein-oligonucleotide conjugates in a 93% yield within fifteen minutes. Using SPR, we explore how the choice of affinity protein, conjugation strategy, and DNA length impact target binding and reveal the deleterious effects of non-specific conjugation methods. Furthermore, we show that these adverse effects can be minimised by employing our site-selective conjugation strategy, leading to improved performance in an immuno-PCR assay. Finally, we investigate the interactions between affinity protein-oligonucleotide conjugates and live cells, demonstrating the benefits of site-selective conjugation. This work provides critical insight into the importance of conjugation strategy when constructing affinity protein-oligonucleotide conjugates. Site-selective conjugation of ssDNA to affinity proteins grants improved target binding and lower non-specific binding.
Bibliography:https://doi.org/10.1039/d4sc01838a
Electronic supplementary information (ESI) available. See DOI
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Both authors contributed equally.
ISSN:2041-6520
2041-6539
DOI:10.1039/d4sc01838a