Orthogonal protein decoration of DNA nanostructures based on SpyCatcher–SpyTag interaction

Kröll, Sandra; Schneider, Leonie; Wadhwani, Parvesh; Rabe, Kersten S.; Niemeyer, Christof M.

doi:10.1039/d2cc05335g

Cited by 7 publications

(10 citation statements)

References 42 publications

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“…The significant difference in k cat of @DON and DON@MB samples further indicated a synergistic effect by immobilization in the compartments of DON and MB, which is consistent with previous observations obtained for comparable but different ligation chemistry based DON@MB‐LbADH systems. [ 40 ] Therein, a SpyCatcher‐modified LbADH showed 40% higher activity than the free enzyme upon immobilization. Thus, the results indicate that the type of immobilization can have a substantial impact on the activity of immobilized enzymes and highlight the importance of a comprehensive characterization of individual enzymes before considering interacting cascades of immobilized enzymes.…”

Section: Resultsmentioning

confidence: 99%

“…Both systems use genetically-encoded fusion tags on the proteins that couple with high efficiency to corresponding suicide ligands on the DON, chlorohexyl (CH) and benzylguanine (BG) ligands for the HOB tag and SNAP-tag, respectively, in a site-specific and orthogonal manner. Here, we chose this approach for immobilizing the enzymes on DON because the high performance of this type of DON decoration had been demonstrated in numerous examples by us [19,[34][35][36][37][38][39][40] and others. [21,[41][42][43] Furthermore, we developed an HPLC method coupled with highly sensitive mass spectrometry (MS) detection to optimize accurate time-resolved quantification of reaction products and gain insights into the reaction-diffusion network effects of spatially organized biocatalytic cascades.…”

Section: Resultsmentioning

confidence: 99%

“…Building on our previous studies on KRED-based DNA origami nanostructures, [35,39,40] we here investigated a cascade consisting of the tetrameric (R)-selective alcohol dehydrogenase LbADH (EC 1. 1.1.2) from Lactobacillus brevis and the dimeric isocitrate dehydrogenase ICDH (EC 1.1.1.42) from Bacillus subtilis.…”

Section: Design and Assembly Of The Experimental Model Systemmentioning

confidence: 99%

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Nano‐ and Microscale Confinements in DNA‐Scaffolded Enzyme Cascade Reactions

Kröll,

Burgahn,

Rabe

et al. 2023

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Artificial reconstruction of naturally evolved principles, such as compartmentalization and cascading of multienzyme complexes, offers enormous potential for the development of biocatalytic materials and processes. Due to their unique addressability at the nanoscale, DNA origami nanostructures (DON) have proven to be an exceptionally powerful tool for studying the fundamental processes in biocatalytic cascades. To systematically investigate the diffusion‐reaction network of (co)substrate transfer in enzyme cascades, a model system of stereoselective ketoreductase (KRED) with cofactor regenerating enzyme is assembled in different spatial arrangements on DNA nanostructures and is located in the sphere of microbeads (MB) as a spatially confining nano‐ and microenvironment, respectively. The results, obtained through the use of highly sensitive analytical methods, Western blot‐based quantification of the enzymes, and mass spectrometric (MS) product detection, along with theoretical modeling, provide strong evidence for the presence of two interacting compartments, the diffusion layers around the microbead and the DNA scaffold, which influence the catalytic efficiency of the cascade. It is shown that the microscale compartment exerts a strong influence on the productivity of the cascade, whereas the nanoscale arrangement of enzymes has no influence but can be modulated by the insertion of a diffusion barrier.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Design and Assembly Of The Experimental Model Systemmentioning

confidence: 99%

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Nano‐ and Microscale Confinements in DNA‐Scaffolded Enzyme Cascade Reactions

Kröll,

Burgahn,

Rabe

et al. 2023

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“…Increasing numbers of functional proteins have been SpyTagged (Hentrich et al, 2021; Keeble and Howarth, 2019; Kellmann et al, 2023), allowing purification (Khairil Anuar et al, 2019; Vester et al, 2022), immobilization (Wang et al, 2020), multimerization (Brune and Howarth, 2018) and networking to other molecular functionalities (Keeble and Howarth, 2020). Beyond proteins, SpyTag/SpyCatcher has been functionalized with nucleic acids (Kröll et al, 2022), oligosaccharides (Li et al, 2022) or small molecules (Geissinger et al, 2020), which will allow modular assembly of bispecifics with more diverse activities. We anticipate applications of SpyCombinator beyond cell signaling, such as enhancing the recruitment of stem cells for tissue repair (Cousens et al, 2009) or optimizing enzyme networks for catalysis (Wei et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

SpyMask Enables Combinatorial Assembly of Bispecific Binders

Driscoll,

Keeble,

Howarth

2023

Preprint

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Bispecific antibodies are a successful and expanding therapeutic class, bridging two cell-types or engaging two different molecules on the same cell. Bispecifics unlock avenues towards synergy, resistance evasion, and signaling bias. Standard approaches to generate bispecifics are complicated by the need for disulfide reduction/oxidation or cloning of each binder molecule in two different formats. Here we present a modular approach to bispecifics using SpyTag/SpyCatcher spontaneous amidation, where all binders are cloned in the same format, bearing a SpyTag. Two SpyTag-fused antigen-binding modules can be precisely conjugated onto DoubleCatcher, a tandem SpyCatcher where the second Catcher is unreactive until unveiling of reactivity using site-specific protease. Assembly on DoubleCatcher is efficient in phosphate-buffered saline at 37 °C, with half-times less than 5 min for both SpyCatcher arms and over 97% bispecific homogeneity. We engineer a panel of DoubleCatchers, locked through disulfide bonds to direct binders to project in different directions from the hub. We establish a generalized methodology for one-pot assembly and purification of bispecifics in 96-well plate format. A panel of Fab, affibody or nanobody binders recognizing different sites on HER2 were coupled to DoubleCatcher, revealing unexpected combinations with anti-proliferative or pro-proliferative activity on HER2-addicted cancer cells. Bispecific activity depended sensitively on both the order of the binders within the assembly and the geometry of DoubleCatcher scaffolds. These findings support the need for straightforward assembly in different formats. SpyCombinator provides a simple and scalable tool to discover synergy in bispecific activity, through modulating receptor organization and geometry.

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“…Low occupancy densities, for example, can result in the individual components of a cascade not being co‐immobilized on the same scaffold, which can distort the interpretation of the data. To distinguish between fully functionalized and partially functionalized nanostructures, methods such as atomic force microscopy (AFM) or transmission electron microscopy (TEM) are commonly used [15,21a,31–32,40] and can be complemented by additional methods such as western blot analysis [21c,32c,d] …”

Section: Advances and Challenges In The Analysis Of Dna‐based Cascadesmentioning

confidence: 99%

Nucleic Acid‐based Enzyme Cascades—Current Trends and Future Perspectives

Kröll,

Niemeyer

2023

Angewandte Chemie

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The natural micro‐ and nanoscale organization of biomacromolecules is a remarkable principle within living cells, allowing for the control of cellular functions by compartmentalization, dimensional diffusion and substrate channeling. In order to explore these biological mechanisms and harness their potential for applications such as sensing and catalysis, molecular scaffolding has emerged as a promising approach. In the case of synthetic enzyme cascades, developments in DNA nanotechnology have produced particularly powerful scaffolds whose addressability can be programmed with nanometer precision. In this minireview, we summarize recent developments in the field of biomimetic multicatalytic cascade reactions organized on DNA nanostructures. We emphasize the impact of the underlying design principles like DNA origami, efficient strategies for enzyme immobilization, as well as the importance of experimental design parameters and theoretical modeling. We show how DNA nanostructures have enabled a better understanding of diffusion and compartmentalization effects at the nanometer length scale, and discuss the challenges and future potential for commercial applications.

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Orthogonal protein decoration of DNA nanostructures based on SpyCatcher–SpyTag interaction

Abstract: We present an efficient and readily applicable strategy for the covalent ligation of proteins to DNA origami by using the SpyCatcher-SpyTag (SC-ST) connector system. This approach showed orthogonality with other...

Cited by 7 publications

References 42 publications

Nano‐ and Microscale Confinements in DNA‐Scaffolded Enzyme Cascade Reactions

Nano‐ and Microscale Confinements in DNA‐Scaffolded Enzyme Cascade Reactions

SpyMask Enables Combinatorial Assembly of Bispecific Binders

Nucleic Acid‐based Enzyme Cascades—Current Trends and Future Perspectives

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