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 Despite growing interest, these probes are typically constructed using outdated, non-selective chemistries, and little has been done to investigate how conjugation to oligonucleotides influences the function of affinity proteins.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.

中文翻译：

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单链 DNA 与亲和蛋白的定点缀合：量化缀合策略的重要性


亲和蛋白-寡核苷酸缀合物越来越多地被探索作为诊断和治疗工具。尽管人们的兴趣日益浓厚，但这些探针通常是使用过时的非选择性化学物质构建的，并且很少有人研究与寡核苷酸的缀合如何影响亲和蛋白的功能。在此，我们报告了一种新颖的位点选择性缀合方法，可在 15 分钟内以 93% 的产率提供亲和蛋白-寡核苷酸缀合物。使用 SPR，我们探索了亲和蛋白的选择、缀合策略和 DNA 长度如何影响靶标结合，并揭示了非特异性缀合方法的有害影响。此外，我们表明，通过采用我们的位点选择性缀合策略可以最大限度地减少这些不利影响，从而提高免疫 PCR 测定的性能。最后，我们研究了亲和蛋白-寡核苷酸缀合物与活细胞之间的相互作用，证明了位点选择性缀合的好处。这项工作为构建亲和蛋白-寡核苷酸缀合物时缀合策略的重要性提供了重要的见解。