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Development of fast-dissociating recombinant antibodies for high-density multiplexed IRIS super-resolution microscopy

Zhang, Qianli 京都大学 DOI:10.14989/doctor.k24304

2022.11.24

概要

Single-molecule localization super-resolution microscopy (SMLM) has greatly surpassed the diffraction limit of conventional optical microscopy. The imaging fidelity, however, is limited by the labeling density of antibodies. Image reconstruction by integrating exchangeable single-molecule localization (IRIS), which uses exchangeable probes that transiently bind to endogenous targets , overcomes the problem. Generation of fast-dissociating IRIS probes has been challenging in the previous studies. Because the number of available antibodies is expanding rapidly, generating recombinant probes from existing antibody sequences may greatly expand the usability of IRIS, but such an approach would require an efficient strategy to accelerate the dissociation of the antibody-target interaction without compromising the binding specificity.

　In this study, the applicant found that mutagenesis at the base of complementarity determining region (CDR) loops may effectively accelerate dissociation of antibodies without loss of binding specificity. The amino acids at these sites are conserved and often occupied by tyrosine residues, which play dominant roles in mediating molecular contacts. By combining multiple site- directed mutagenesis at these conserved sites, a versatile strategy that can rapidly convert the existing antibody cDNAs to fast-dissociating fluorescent probes for IRIS multiplexed super-resolution imaging was developed.

　The applicant successfully generated dozens of fast-dissociating antibody- derived IRIS probes and demonstrate multiplexed localization of endogenous proteins in primary neurons that visualizes synaptic connections as small as ~0.1 μm. The average binding events of IRIS probes against Homer and VGLUT in individual synapse were 694 and 3798, respectively. Because the number of Homer and VGLUT molecules in a synapse are reported to be 233 and 8254, approximately 95% of Homer and 37% of VGLUT molecules were estimated to be labeled at least once.

　In addition, the applicant shows evidence of the spatial interference between multiple antibodies in small synapse areas, which could give rise to sparse labeling in conventional SMLM such as stochastic optical reconstruction microscopy (STORM) and DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT). Fast-dissociating IRIS probes do not suffer from this problem. Furthermore, multiple localization points may arise from repeated detection of a single label in STORM and DNA-PAINT. IRIS thus achieves higher label density than conventional super-resolution approaches, and visualizes features of synaptic components with higher fidelity.

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