Abstract:Site-specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self-quenching of multiple fluorophores installed in close proximity, we used Holliday junctions to label proteins site-specifically. Holliday junctions enable modification with multiple fluorophores at reasonably precise spacing. We designed a Holliday junction with three of its four arms modified with a fluorophore of choice and the remaining arm equipped with a dibenzocyclooc… Show more
“…G 3 -Biotin, G 3 -TMR, or G 3 -Alexa647 were synthesized, as described in refs 14, 37, and 38. For feeding, nucleophiles in water were diluted to a final concentration of 1 mM in NGM containing living OP50 E. coli bacteria as food-source L4 stage larvae or young adult nematodes were added, and animals were incubated for 5 h at RT with gentle shaking.…”
In vivo protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA7m) expressed in C. elegans is functional and supports in vitro sortase reactions in a low-Ca2+ environment. Feeding SrtA7m-expressing C. elegans with small peptide-based probes such as (Gly)3- biotin or (Gly)3-fluorophores enables in vivo target protein modification. SrtA7m also catalyzes the circularization of suitably modified linear target proteins in vivo and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve in vivo protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins in vivo.
“…G 3 -Biotin, G 3 -TMR, or G 3 -Alexa647 were synthesized, as described in refs 14, 37, and 38. For feeding, nucleophiles in water were diluted to a final concentration of 1 mM in NGM containing living OP50 E. coli bacteria as food-source L4 stage larvae or young adult nematodes were added, and animals were incubated for 5 h at RT with gentle shaking.…”
In vivo protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA7m) expressed in C. elegans is functional and supports in vitro sortase reactions in a low-Ca2+ environment. Feeding SrtA7m-expressing C. elegans with small peptide-based probes such as (Gly)3- biotin or (Gly)3-fluorophores enables in vivo target protein modification. SrtA7m also catalyzes the circularization of suitably modified linear target proteins in vivo and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve in vivo protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins in vivo.
“…Specific binders are easily selected, manipulated and produced in large amounts using standard recombinant techniques. Expectedly, a continuously growing array of applications in research, diagnostics and therapy emerged in recent years that uses nanobodies in cell biology 8 , structural biology 9 , for super-resolution microscopy 10 , 11 , as diagnostic agents 12 or potent inhibitors of cancer-associated proteins 13 . Figure 1 Parameters determined in the nanobody analysis.…”
Nanobodies represent the variable binding domain of camelid heavy-chain antibodies and are employed in a rapidly growing range of applications in biotechnology and biomedicine. Their success is based on unique properties including their reported ability to reversibly refold after heat-induced denaturation. This view, however, is contrasted by studies which involve irreversibly aggregating nanobodies, asking for a quantitative analysis that clearly defines nanobody thermoresistance and reveals the determinants of unfolding reversibility and aggregation propensity. By characterizing nearly 70 nanobodies, we show that irreversible aggregation does occur upon heat denaturation for the large majority of binders, potentially affecting application-relevant parameters like stability and immunogenicity. However, by deriving aggregation propensities from apparent melting temperatures, we show that an optional disulfide bond suppresses nanobody aggregation. This effect is further enhanced by increasing the length of a complementarity determining loop which, although expected to destabilize, contributes to nanobody stability. The effect of such variations depends on environmental conditions, however. Nanobodies with two disulfide bonds, for example, are prone to lose their functionality in the cytosol. Our study suggests strategies to engineer nanobodies that exhibit optimal performance parameters and gives insights into general mechanisms which evolved to prevent protein aggregation.
“…This represents a significant improvement compared to other nucleic acid-based platforms with a potential use for drug delivery, such as doxorubicin-loaded DNA origami structures 46 that risk dissociation and drug leakage at the physiological salt concentrations and low concentrations in vivo 47. Due to its small size and highly chemically modified nature, the HJ presented here also separates itself from similar proposed structures, such as a significantly larger DNA HJ consisting of four 50 nt long DNA strands, all modified with fluorophores for high-signal protein labeling 48.…”
Rationale
: Within the field of personalized medicine there is an increasing focus on designing flexible, multifunctional drug delivery systems that combine high efficacy with minimal side effects, by tailoring treatment to the individual.
Methods
: We synthesized a chemically stabilized ~4 nm nucleic acid nanoscaffold, and characterized its assembly, stability and functional properties
in vitro
and
in vivo
. We tested its flexibility towards multifunctionalization by conjugating various biomolecules to the four modules of the system. The pharmacokinetics, targeting capability and bioimaging properties of the structure were investigated in mice. The role of avidity in targeted liver cell internalization was investigated by flow cytometry, confocal microscopy and
in vivo
by fluorescent scanning of the blood and organs of the animals.
Results
: We have developed a nanoscaffold that rapidly and with high efficiency can self-assemble four chemically conjugated functionalities into a stable,
in vivo
-applicable system with complete control of stoichiometry and site specificity. The circulation time of the nanoscaffold could be tuned by functionalization with various numbers of polyethylene glycol polymers or with albumin-binding fatty acids. Highly effective hepatocyte-specific internalization was achieved with increasing valencies of tri-antennary galactosamine (triGalNAc)
in vitro
and
in vivo
.
Conclusion
: With its facile functionalization, stoichiometric control, small size and high serum- and thermostability, the nanoscaffold presented here constitutes a novel and flexible platform technology for theranostics.
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