Engineered fast-dissociating antibody fragments for multiplexed super-resolution microscopy

Superresolution Imaging with Single-Antibody Labeling

“…Limitations of the technique include the extended image acquisition and relatively low labeling densities due to the slow off-rate of the full-length antibodies. Our previous work showed antibody fragmentation could result in faster dissociation, while others have utilized protein engineering approaches. , Nevertheless, promoting antibody dissociation without compromising its binding specificity is likely a complex task and highly dependent upon the nature of antibody–antigen interactions.…”

“…Our previous work showed antibody fragmentation could result in faster dissociation, 8 while others have utilized protein engineering approaches. 14,15 Nevertheless, promoting antibody dissociation without compromising its binding specificity is likely a complex task and highly dependent upon the nature of antibody−antigen interactions. Unlike standard IF staining procedures, which remove nonspecifically bound antibodies through the washing steps, time-lapse imaging of single-antibody labeling captures both specific and nonspecific events.…”

“…8 More recently, similar strategies to enable transient protein-based interactions for PAINT-based superresolution imaging have been reported. These techniques include universal-PAINT (u-PAINT) employing labeled ligands and antibodies to label tagged and endogenous membrane proteins, 9 peptide-PAINT using coiled-coil interactions, 10 peptide-PAINT using docking peptides, 11,12 protein-PAINT (pPAINT) using signaling proteins to investigate Tcell signaling with multiplexing capability, 13 fast-dissociating antibodies generated through hybridoma technology and their fragments, 14 and engineered fast-dissociating antibody fragments 15 generated via site-specific mutations against commonly used molecular epitope tags. While these techniques represent significant technical advancements, a more versatile approach remains desirable for using readily available antibodies.…”

When Weak Is Strong: A Plea for Low‐Affinity Binders for Optical Microscopy

“…Targeted mutagenesis of antibodies allows increasing the dissociation rate without compromising the specificity, and by that, 'reprogramming' conventional antibodies into exchangeable labels. [19] Protein tags are another excellent option to genetically target proteins, with the additional benefits of live-cell imaging and stoichiometric protein labeling. For example, engineered mutants of the bacterial lipocalin Blc that bind a cell-permeable fluorogenic dye enabled live-cell fluorescence microscopy with a renewable signal.…”

Beyond DNA: new probes for PAINT super-resolution microscopy

“…Unfortunately, this technique might not be applicable to all antibodies, as the development requires sequence information and cocrystal structures, which are often lacking in antibodies. 43 Aptamers and engineered antibodies might pave the way to a more rational development of probes, even for endogenous proteins that have an affinity outside of the typical PAINT regime. However, screening for new suitable probes with the correct affinity is labour intensive.…”