Turning Proteins into Assays:Affinity-Guided Bioconjugation in the Assembly of Continuous Sensors

Continuous monitoring of small-molecule analytes is critical for advancing personalised medicine, enabling the tracking of biomarkers and therapeutic drug levels in real-time. Current diagnostic assays, based on batch measurements, offer only snapshot information and are insufficient for capturing dynamic biological processes. This has created a demand for novel assay technologies capable of continuous detection and quantification of target analytes.<br/>This PhD dissertation focuses on the development of a modular platform for continuous monitoring of small-molecule analytes using a DNA-protein nanoswitch, described at the end of Chapter 1. The aim was to create a versatile assay system that could be adapted for different target analytes, beginning with a digoxigenin-based assay as a proof of concept.<br/>The critical step in assembling this assay was an affinity-guided DNA-protein conjugation, which is investigated in Chapter 2. For this approach, we designed a DNA scaffold and selectively modified it with a guiding moiety and custom linker molecules. Both modifications were optimised to bring DNA close to the binding site of the protein and therefore label adjacent lysine residues in a site-directed manner. To create a covalent bond between the two biomolecules, we explored and optimised two different conjugation chemistries: reductive amination and amide coupling reactions. Using both techniques, we were able to obtain and purify the desired DNA-protein conjugate.<br/>Once the conjugate was acquired, it was integrated with an optical readout system to create a functional assay, as described in Chapter 3. The modular DNA scaffold design allowed it to be paired with a variety of reporters through strand hybridisation. We explored the Cy3/Cy5 FRET pair and demonstrated successful incorporation of the dyes, as well as low-nanomolar dose-responsive behaviour when incubating the assay with digoxigenin. Furthermore, we investigated the potential to fine-tune the assay’s kinetics and dynamic range by modifying the length of the complementary strands.<br/>In Chapter 4, the assay's potential for continuous monitoring was investigated by integrating the DNA-protein nanoswitch into different sensing systems. Firstly, biolayer interferometry experiments yielded promising results, showing real-time assay interaction with digoxigenin and its release. Afterwards, we looked into immobilisation techniques using glass or polymer capsules. The immobilised assay was then integrated into a flow-through channel system and interrogated with an epifluorescence setup, demonstrating reversible behaviour.<br/>Chapter 5 examined the versatility and limitations of assay design. Extending the system to other small molecules – terephthalic acid (TPA) and cortisol – revealed both the platform's adaptability and constrains. While the DNA scaffold could be universal to multiple targets, a highly specific TPA-recognising protein exhibited binding site obstructions that compromised the affinity-guided approach. In contrast, full-antibody-based assays for cortisol had successful bioconjugation but demonstrated the challenge of optimising conjugates with dual binding sites.<br/>Lastly, the thesis ends with a conclusion and an epilogue that summarises the results presented, the work from side projects, and some personal reflections.