A self-assembling protein–DNA complex with an inbuilt DNA release system for quantitative immuno-PCR applications

Abstract

Site-specific protein : DNA conjugation is gaining increasing importance in detection technologies such as quantitative immuno-PCR (qIPCR). Until now, DNA-binding proteins have been a relatively untapped source of protein : DNA conjugation systems. In Escherichia coli, the biotin protein ligase (BirA) is a biotin-dependent DNA-binding protein that offers a means to connect a protein of interest (POI) with DNA. Here, we explored BirA as a unique on–off protein : DNA connection switch for the production of self-assembling POI : DNA conjugates. Green fluorescent protein (GFP) is a versatile protein tag and reporter, commonly quantified by fluorescence detection. However, low GFP concentrations are challenging to detect and require more sensitive methods. A multitude of high-affinity antibodies are available for capture and detection of GFP as an affinity tag. As such, a well-characterised GFP-tagged BirA (BirA-GFP) was selected for the development and validation of an innovative qIPCR platform technology. The unique principle of this assay involves the assembly of two BirA-GFP with the bioO repressor DNA sequence in the presence of ATP and biotin. The resulting high affinity bioO : BirA-GFP complex can be applied in various formats to detect the presence of anti-GFP IgG as well as GFP immobilised on a surface. Complete release of the quantifiable bioO DNA can easily be achieved by omitting ATP and biotin in the final elution step. The new BirA-based qIPCR assay enabled picomolar (≥10⁻¹² M) detection of GFP and anti-GFP IgG as well as their affinity profiling.