Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox.

Peptide tags are a key resource, introducing minimal change while enabling a consistent process to purify diverse proteins. However, peptide tags often provide minimal benefit post-purification. We previously designed SpyTag, forming an irreversible bond with its protein partner SpyCatcher. SpyTag provides an easy route to anchor, bridge or multimerize proteins. Here we establish Spy&Go, enabling protein purification using SpyTag. Through rational engineering we generated SpyDock, which captures SpyTag-fusions and allows efficient elution. Spy&Go enabled sensitive purification of SpyTag-fusions from Escherichia coli, giving superior purity than His-tag/nickel-nitrilotriacetic acid. Spy&Go allowed purification of mammalian-expressed, N-terminal, C-terminal or internal SpyTag. As an oligomerization toolbox, we established a panel of SpyCatcher-linked coiled coils, so SpyTag-fusions can be dimerized, trimerized, tetramerized, pentamerized, hexamerized or heptamerized. Assembling oligomers for Death Receptor 5 stimulation, we probed multivalency effects on cancer cell death. Spy&Go, combined with simple oligomerization, should have broad application for exploring multivalency in signaling.

Nanoteamwork: covalent protein assembly beyond duets towards protein ensembles and orchestras.

Biological processes often depend on the harmonious interplay of multiple macromolecules. Biotechnology has had great success in applying and modifying individual components, but the building of multi-component teams is at an early stage. Cells are intelligent in sensing their environment, so manipulating just one signal can limit potency and promote side-effects for therapeutics. Here we critically assess the latest advances in irreversibly connecting individual protein units, through different spontaneous or catalysed reactions. Then we outline efforts to go beyond bipartite assembly, towards multimeric or sequence-programmed architectures. These early steps will be put in context of the enormous opportunities for synthetic protein nanomachines, focusing on catalysis and the control of cell signalling for cancer and the immune system.

Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics.

SpyTag is a peptide that forms a spontaneous amide bond with its protein partner SpyCatcher. This protein superglue is a broadly useful tool for molecular assembly, locking together biological building blocks efficiently and irreversibly in diverse architectures. We initially developed SpyTag and SpyCatcher by rational design, through splitting a domain from a Gram-positive bacterial adhesin. In this work, we established a phage-display platform to select for specific amidation, leading to an order of magnitude acceleration for interaction of the SpyTag002 variant with the SpyCatcher002 variant. We show that the 002 pair bonds rapidly under a wide range of conditions and at either protein terminus. SpyCatcher002 was fused to an intimin derived from enterohemorrhagic Escherichia coli. SpyTag002 reaction enabled specific and covalent decoration of intimin for live cell fluorescent imaging of the dynamics of the bacterial outer membrane as cells divide.

A novel type of quantum dot-transferrin conjugate using DNA hybridization mimics intracellular recycling of endogenous transferrin.

Colloidal nanoparticles such as Quantum Dots (QDs) are promising alternatives to organic fluorophores, especially for long duration bioimaging. For specific targeting applications, QDs frequently require functionalization with selected proteins. In this regard, conjugation of proteins to QDs such that the nanobioconjugates retain the endogenous behavior of the coupled protein remains challenging. We have developed a novel method to conjugate a protein, transferrin (Tf), to QDs using DNA hybridization. These conjugates are characterized biochemically, and the trafficking properties in live cells are investigated. Although the internalization kinetics into the cells is much reduced compared to Tf labelled with organic dye, we could show that DNA hybridization-based QD-Tf conjugates are the first for which recycling from endosomes to the plasma membrane can be observed. This recycling occurs with kinetics that is similar to dye labelled Tf. We could image and follow the trajectories of recycling of individual vesicles for several tens of minutes. The conjugation of QDs to proteins mediated by DNA hybridization yields a new generation of ultra-bright and photostable probes that preserves the intracellular properties of the dye labelled protein better than previously reported QD conjugates using other surface chemistries for direct coupling.

Quantum dots-DNA bioconjugates: synthesis to applications.

Semiconductor nanoparticles particularly quantum dots (QDs) are interesting alternatives to organic fluorophores for a range of applications such as biosensing, imaging and therapeutics. Addition of a programmable scaffold such as DNA to QDs further expands the scope and applicability of these hybrid nanomaterials in biology. In this review, the most important stages of preparation of QD-DNA conjugates for specific applications in biology are discussed. Special emphasis is laid on (i) the most successful strategies to disperse QDs in aqueous media, (ii) the range of different conjugation with detailed discussion about specific merits and demerits in each case, and (iii) typical applications of these conjugates in the context of biology.

Fast, Efficient, and Stable Conjugation of Multiple DNA Strands on Colloidal Quantum Dots.

A novel method for covalent conjugation of DNA to polymer coated quantum dots (QDs) is investigated in detail. This method is fast and efficient: up to 12 DNA strands can be covalently conjugated per QD in optimized reaction conditions. The QD-DNA conjugates can be purified using size exclusion chromatography and the QDs retain high quantum yield and excellent stability after DNA coupling. We explored single-stranded and double-stranded DNA coupling, as well as various lengths. We show that the DNA coupling is most efficient for short (15 mer) single-stranded DNA. The DNA coupling has been performed on QDs emitting at four different wavelengths, as well as on gold nanoparticles, suggesting that this technique can be generalized to a wide range of nanoparticles.