Fig. 4. Enzyme resilience and connection using Spy technology. (A) Cyclisation with SpyTag/SpyCatcher can increase enzyme resilience. SpyTag-β-lactamase-SpyCatcher forms an intramolecular isopeptide bond. Upon heating at the indicated temperature for 10 min, aggregated protein was removed by centrifugation and soluble fraction determined (mean ± 1 s.d., n = 3; adapted from ref. 63). (B) All-enzyme hydrogel. Mixing SpyTag-tetrameric enzyme with SpyCatcher-dimeric enzyme leads to rapid gelation and stable catalytic function. (C) Assisted substrate recognition. Sortase-SpyCatcher efficiently recruits SpyTag-linked substrate, with release triggered by an oligoglycine-linked biophysical probe. (D) Next-generation sequencing. SpyTag can anchor enzymes to nanopores, with single-molecule detection of DNA sequence from the effect of the tail-modified deoxynucleoside triphosphate (dNTP) on the current.