Protein-DNA Conjugates with a Discrete Number of Oligonucleotide Strands for Highly Reproducible Protein Quantification by the DNA Proximity Assay.
Anal Chem
; 95(32): 12071-12079, 2023 08 15.
Article
en En
| MEDLINE
| ID: mdl-37523447
Protein-oligonucleotide conjugates are increasingly used as detection probes in biological applications such as proximity sensing and spatial biology. The preparation of high-quality conjugate probes as starting reagents is critical for achieving good and consistent performance, which we demonstrate via the DNA proximity assay (DPA) for the one-pot quantification of protein targets. We first established a complete conjugation and anion-exchange chromatography purification workflow to reproducibly obtain pure subpopulations of protein probes carrying a discrete number of oligonucleotide strands. A systematic study using the purified conjugate sub-populations confirmed that the order of conjugate (number of oligonucleotides per protein) and its purity (the absence of the unconjugated antibody) were important for ensuring optimal and reproducible assay performance. The streamlined workflow was then successfully used to conjugate a pair of universal DPA initiator oligonucleotides onto a wide range of binders including antibodies, nanobodies, and antigens which enabled the versatile detection of different types of proteins such as cytokines, total antibodies, and specific antibody isotypes. The good assay robustness (the inter-assay coefficient of variation lower than 5%) and linear calibration curve was achieved across all targets with just a single mix-and-incubate reaction step and a short reaction time of 30 min. We anticipate the streamlined protein-oligonucleotide probe preparation workflow developed in this work to have broad utility across applications leveraging the specificity of protein bio-recognition with the programmability of DNA hybridization.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Oligonucleótidos
/
ADN
Idioma:
En
Revista:
Anal Chem
Año:
2023
Tipo del documento:
Article
País de afiliación:
Singapur
Pais de publicación:
Estados Unidos