ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces

AbstractssDNA recombineering has been exploited to hyperdiversify genomically-encoded nanobodies displayed on the surface of Escherichia coli for originating new binding properties. As a proof-of-principle a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) was evolved towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC). To this end, E. coli cells displaying this nanobody fused to the intimin outer membrane-bound domain were subjected to multiple rounds of mutagenic oligonucleotide recombineering targeting the complementarity determining regions (CDRs) of the cognate VHH gene sequence. Binders to the EPEC-TirM were selected upon immunomagnetic capture of bacteria bearing active variants and nanobodies identified with a new ability to strongly bind the new antigen. The results highlight the power of combining evolutionary properties of bacteria in vivo with oligonucleotide synthesis in vitro for the sake of focusing diversification to specific segments of a gene (or protein thereof) of interest.

Exosomal Surface Protein Detection with Quantum Dots and Immunomagnetic Capture for Cancer Detection

Exosomes carry molecular contents reflective of parental cells and thereby hold great potential as a source of biomarkers for non-invasive cancer detection and monitoring. However, simple and rapid exosomal molecular detection remains challenging. Here, we report a facile method for exosome surface protein detection using quantum dot coupled with immunomagnetic capture and enrichment. In this method, exosomes were captured by magnetic beads based on CD81 protein expression. Surface protein markers of interest were recognized by primary antibody and then detected by secondary antibody-conjugated quantum dot with fluorescent spectroscopy. Validated by ELISA, our method can specifically detect different surface markers on exosomes from different cancer cell lines and differentiate cancer exosomes from normal exosomes. The clinical potential was demonstrated with pilot plasma samples using HER2-positive breast cancer as the disease model. The results show that exosomes from HER2-positive breast cancer patients exhibited a five times higher level of HER2 expression than healthy controls. Exosomal HER2 showed strong diagnostic power for HER2-positive patients, with the area under the curve of 0.969. This quantum dot-based exosome method is rapid (less than 5 h) and only requires microliters of diluted plasma without pre-purification, practical for routine use for basic vesicle research, and clinical applications.

ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces

SUMMARYIn vivo evolution of antibodies facilitates emergence of novel target specificities from pre-existing clones. In this work we show how mutagenic ssDNA recombineering of camel-derived nanobodies encoded in a bacterial genome enables clonal hyper-diversification and the rise of new properties. As a proof-of-principle we used a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) and evolved it towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC). To this end, E. coli cells displaying on their surface this nanobody fused to the intimin outer membrane anchor domain were subjected to multiple rounds of mutagenic ssDNA recombineering targeted to the CDR1, CDR2 and CDR3 regions of its genomically encoded VHH sequence. Binders to the new antigen (EPEC TirM) were then selected upon immunomagnetic capture of bacteria bearing the corresponding nanobody variants. As a result, several modified nanobodies were identified which maintained recognition of EHEC TirM but acquired the ability to bind the new antigen with high affinity (Kd ~20 nM). The results highlight the power of combining evolutionary properties of bacteria in vivo with oligonucleotide synthesis in vitro for the sake of focusing diversification to specific segments of a gene (or protein thereof) of interest. Our experimental workflow empowers the evolution of nanobodies displayed on the surface of bacterial cells for a large number of potential applications in medical and industrial biotechnology.

Polymer-based microfluidic chip for rapid and efficient immunomagnetic capture and release of Listeria monocytogenes

A 3D magnetic trap is integrated on a polymeric microfluidic device using rapid low-cost fabrication. The device is used for efficient magnetic capture and release of bacteria conjugated to immunomagnetic nanoparticles.