Oligonucleotide-based biosensors, increasingly used in immunosensors, have diverse applications, especially in in vitro protein diagnostics. We are working on a DNA-based biosensor to detect antibody-antigen interaction at the single molecule level. Using a single-chain variable fragment (scFv) against the model antigen MBP, we create an ‘oligobody’ by attaching complementary oligonucleotides to the scFv. In the absence of antigen, intra-oligo hybridization occurs, while the presence of antigen blocks this process. This allows us to detect antigen binding by introducing a replenishable DNA probe that binds to the free oligo 'arm.
Single-molecule TIRF (SM TIRF) microscopy was employed to validate a range of oligonucleotide pairs with varying sequence composition and length that are surface immobilised. Binding kinetics of DNA‑probe hybridisation to one of the oligo ‘arm’ in the presence and absence of a competitor ‘arm’ were compared which mimics intra-oligo hybridisation behaviour in the absence and presence of antigen, respectively. Computational simulations suggest short (<5 bp) strands allow intra-oligo hybridisation over the antigen-binding interface with minimal sampling onto the surroundings. Using cell-free protein production machinery, we obtain a functional 'oligobody' which binds to the cognate antigen, MBP. Palindromic intra-oligos with varying lengths were conjugated on the periphery of scFv, while unnatural amino acid incorporation was utilised to site-specifically conjugate dual-distinct oligos.
Preliminary SM TIRF analysis suggest increased probe binding in the presence of antigen, more prevalent with short intra-oligo pairs (4bp) compared to longer pairs (6, 8bp). Strategies to increase the fidelity of DNA-probe hybridisation in the presence of antigen, while minimising probe binding in the absence is necessary to increase signal-to-noise ratio. Future studies will utilise shorter DNA-probe with lower binding free energy compared to intra-oligo hybridisation. Detection of antigen binding by DNA-probe binding kinetic signatures will underlie further development of oligobodies against clinically relevant biomolecular targets.