Light-guided intrabodies for on-demand <i>in situ</i> target recognition in human cells

Joest, Eike F.; Wesalo, Joshua S.; Deiters, Alexander; Tampé, Robert

doi:10.1039/d1sc01331a

Cited by 16 publications

(25 citation statements)

References 51 publications

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“…We show that this switch in binding affinity is in a useful range for cell culture experiments when addressing the ALFA‐tag as an extracellular epitope. Although not reported here, we and others have previously demonstrated that nanobodies photocaged with the same chemistry of the ONBY‐approach can be activated when binding to or when being located inside mammalian cells, by directly irradiating the cells for a few seconds, thus suggesting the same to be feasible for the ALFA‐Pb [2,3b,25] . The ALFA‐Pb represents the sixth example of a successful photobody design that so far only required a properly positioned tyrosine in the paratope region, thereby underlining the simplicity and generality of the approach.…”

Section: Discussionmentioning

confidence: 77%

A Light‐Activatable Photocaged Variant of the Ultra‐High Affinity ALFA‐Tag Nanobody

Jedlitzke

Mootz

2022

ChemBioChem

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Nanobodies against short linear peptide‐epitopes are widely used to detect and bind proteins of interest (POI) in fusion constructs. Engineered nanobodies that can be controlled by light have found very recent attention for various extra‐ and intracellular applications. We here report the design of a photocaged variant of the ultra‐high affinity ALFA‐tag nanobody, also termed ALFA‐tag photobody. ortho‐Nitrobenzyl tyrosine was incorporated into the paratope region of the nanobody by genetic code expansion technology and resulted in a ≥9,200 to 100,000‐fold impairment of the binding affinity. Irradiation with light (365 nm) leads to decaging and reconstitutes the native nanobody. We show the light‐dependent binding of the ALFA‐tag photobody to HeLa cells presenting the ALFA‐tag. The generation of the first photobody directed against a short peptide epitope underlines the generality of our photobody design concept. We envision that this photobody will be useful for the spatiotemporal control of proteins in many applications using cultured cells.

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Section: Discussionmentioning

confidence: 77%

A Light‐Activatable Photocaged Variant of the Ultra‐High Affinity ALFA‐Tag Nanobody

Jedlitzke

Mootz

2022

ChemBioChem

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“…[32] Furthermore, photo-caging of eNB using ONBY or NPY resulted in blocking of target binding when nanobody and target were coexpressed in cultured HeLa cells. [36,37] While ONBY was not sufficient to abolish binding in our intracellular assay, we found that uncaging by illuminating animals using a 365 nm LED (640 seconds illumination, 10 mW/…”

Section: Npy and Onby Substitution Is Not Sufficient To Photocage Ant...mentioning

confidence: 76%

“…[32,34] Intracellular use of photo-caged nanobodies was first shown by delivering purified photocaged nanobodies synthesised in E. coli to HeLa cells expressing the antigen, [35] and subsequently by directly expressing a photo-caged nanobody in a cultured human cell line. [36,37] A number of factors influence nanobody/antigen interaction, including pH, ionic concentration, incubation time, and concentration of nanobody and antigen. [38] The disruption of binding by the photocaged amino acid observed in one system may therefore not correctly predict binding when applying photocaged nanobodies in other systems or at different concentrations.…”

Section: Introductionmentioning

confidence: 99%

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Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering**

et al. 2022

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Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation using 365 nm light, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies that function robustly when directly expressed in a complex intracellular environment together with their antigen. We apply them to control subcellular protein localisation in the nematode worm Caenorhabditis elegans. Our approach applies predictions derived from computational modelling directly in a living animal and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.

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“…Incorporation of photocaged amino acids into nAbs has also been used to generate optobodies (Joest et al 2021). An alternative approach is to use chemical or light stimulation to induce the coupling of an intrabody to a specific functional protein, in this case an E3 ubiquitin ligase, to yield stimulus-dependent target protein degradation (Deng et al 2020).…”

Section: Summary and Future Perspectivesmentioning

confidence: 99%

Controlling ion channel function with renewable recombinant antibodies

Colecraft

Trimmer

2022

The Journal of Physiology

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Selective ion channel modulators play a critical role in physiology in defining the contribution of specific ion channels to physiological function and as proof of concept for novel therapeutic strategies. Antibodies are valuable research tools that have broad uses including defining the expression and localization of ion channels in native tissue, and capturing ion channel proteins for subsequent analyses. In this review, we detail how renewable and recombinant antibodies can be used to control ion channel function. We describe the different forms of renewable and recombinant antibodies that have been used and the mechanisms by which they modulate ion channel function. We highlight the use of recombinant antibodies that are expressed intracellularly (intrabodies) as genetically encoded tools to control ion channel function. We also offer perspectives of avenues of future research that may be opened by the application of emerging technologies for engineering recombinant antibodies for enhanced utility in ion channel research. Overall, this review provides insights that may help stimulate and guide interested researchers to develop and incorporate renewable and recombinant antibodies as valuable tools to control ion channel function.

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Light-guided intrabodies for on-demand in situ target recognition in human cells

Abstract: Nanobodies are ideal to visualize and modulate targets in living cells. We designed a versatile platform for generating photo-conditional intrabodies by genetic code expansion. After illumination, the intrabodies show fast and stable binding.

Cited by 16 publications

References 51 publications

A Light‐Activatable Photocaged Variant of the Ultra‐High Affinity ALFA‐Tag Nanobody

A Light‐Activatable Photocaged Variant of the Ultra‐High Affinity ALFA‐Tag Nanobody

Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering**

Controlling ion channel function with renewable recombinant antibodies

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