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The delivery of functional molecules to the cell nucleus enables the visualization of nuclear function and the development of effective medical treatments. In this study, we successfully modified the Hoechst molecule, which is a well-documented nuclear-staining agent, using the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. We prepared Hoechst derivatives bearing an azide group (Hoe-N3) and characterized their SPAAC reactions in the presence of corresponding molecules with a dibenzylcyclooctyne unit (DBCO). The SPAAC reaction of Hoe-N3 with alkylamine bearing DBCO, fluorescent TAMRA, or Cy5 molecules bearing DBCO led to the formation of the coupling products Hoe-Amine, Hoe-TAMRA, and Hoe-Cy5, respectively. These Hoechst derivatives retained their DNA-binding properties. In addition, Hoe-TAMRA and Hoe-Cy5 exhibited properties of dual accumulation in the cell nucleus and mitochondria. Initial incubation of these molecules in living cells resulted in its accumulation in mitochondria, while after mitochondrial depolarization, it was smoothly released from mitochondria and translocated into the cell nucleus. Thus, mitochondrial depolarization could be monitored by measuring the emission of Hoe-TAMRA and Hoe-Cy5 at the cell nucleus.
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We describe practical methods to prepare DOTAGA-DBCO and DFO-DBCO from commercially available starting materials. DOTAGA-DBCO is available in five steps from cyclen with a 33 % overall yield at gram scale. Our synthesis of DFO-DBCO also proceeds in five steps from commercially available starting materials. These bifunctional molecules possess chelating functionality for the binding of medically important radiometals and a strained alkyne suitable for Huisgen cyclization with an azide. These syntheses represent an important step toward improved radioimmunoconjugates for imaging and therapeutic applications.
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Ciclamas , Inmunoconjugados , Alquinos/química , Azidas/química , Ciclización , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéuticoRESUMEN
Cross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to substochiometric reaction efficiencies, cross-linked peptides are usually low abundance. This results in challenging data evaluation and the need for an effective enrichment. Here we describe an improved, easy to implement, one-step method to enrich azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) cross-linked peptides using dibenzocyclooctyne (DBCO) coupled Sepharose beads. We probed this method using recombinant Cas9 and E. coli ribosome. For Cas9, the number of detectable cross-links was increased from â¼100 before enrichment to 580 cross-links after enrichment. To mimic a cellular lysate, E. coli ribosome was spiked into a tryptic HEK background at a ratio of 1:2-1:100. The number of detectable unique cross-links was maintained high at â¼100. The estimated enrichment efficiency was improved by a factor of 4-5 (based on XL numbers) compared to enrichment via biotin and streptavidin. We were still able to detect cross-links from 0.25 µg cross-linked E. coli ribosomes in a background of 100 µg tryptic HEK peptides, indicating a high enrichment sensitivity. In contrast to conventional enrichment techniques, like SEC, the time needed for preparation and MS measurement is significantly reduced. This robust, fast, and selective enrichment method for azide-tagged linkers will contribute to mapping protein-protein interactions, investigating protein architectures in more depth, and helping to understand complex biological processes.
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Azidas , Escherichia coli , Reactivos de Enlaces Cruzados , Escherichia coli/genética , Péptidos , SulfóxidosRESUMEN
Molecular tethering of a single membrane protein between the glass surface and a magnetic bead is essential for studying the structural dynamics of membrane proteins using magnetic tweezers. However, the force-induced bond breakage of the widely-used digoxigenin-antidigoxigenin tether complex has imposed limitations on its stable observation. In this chapter, we describe the procedures of constructing highly stable single-molecule tethering methods for membrane proteins. These methods are established using dibenzocyclooctyne click chemistry, traptavidin-biotin binding, SpyCatcher-SpyTag conjugation, and SnoopCatcher-SnoopTag conjugation. The molecular tethering approaches allow for more stable observation of structural transitions in membrane proteins under force.
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Proteínas de la Membrana , NanotecnologíaRESUMEN
The utilization of cytosolic protein delivery is a promising approach for treating various diseases by replacing dysfunctional proteins. Despite the development of various nanoparticle-based intracellular protein delivery methods, the complicated chemical synthesis of the vector, loading efficiency and endosomal escape efficiency of proteins remain a great challenge. Recently, 9-fluorenylmethyloxycarbonyl (Fmoc)-modified amino acid derivatives have been used to self-assemble into supramolecular nanomaterials for drug delivery. However, the instability of the Fmoc group in aqueous medium restricts its application. To address this issue, the Fmoc ligand neighboring arginine was substituted for dibenzocyclooctyne (DBCO) with a similar structure to Fmoc to obtain stable DBCO-functionalized L-arginine derivative (DR). Azide-modified triethylamine (crosslinker C) was combined with DR to construct self-assembled DRC via a click chemical reaction for delivering various proteins, such as BSA and saporin (SA), into the cytosol of cells. The hyaluronic-acid-coated DRC/SA was able to not only shield the cationic toxicity, but also enhance the intracellular delivery efficiency of proteins by targeting CD44 overexpression on the cell membrane. The DRC/SA/HA exhibited higher growth inhibition efficiency and lower IC50 compared to DRC/SA toward various cancer cell lines. In conclusion, DBCO-functionalized L-arginine derivative represents an excellent potential vector for protein-based cancer therapy.
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Single-molecule tweezers, such as magnetic tweezers, are powerful tools for probing nm-scale structural changes in single membrane proteins under force. However, the weak molecular tethers used for the membrane protein studies have limited the observation of long-time, repetitive molecular transitions due to force-induced bond breakage. The prolonged observation of numerous transitions is critical in reliable characterizations of structural states, kinetics, and energy barrier properties. Here, we present a robust single-molecule tweezer method that uses dibenzocyclooctyne cycloaddition and traptavidin binding, enabling the estimation of the folding 'speed limit' of helical membrane proteins. This method is >100 times more stable than a conventional linkage system regarding the lifetime, allowing for the survival for ~12 hr at 50 pN and ~1000 pulling cycle experiments. By using this method, we were able to observe numerous structural transitions of a designer single-chained transmembrane homodimer for 9 hr at 12 pN and reveal its folding pathway including the hidden dynamics of helix-coil transitions. We characterized the energy barrier heights and folding times for the transitions using a model-independent deconvolution method and the hidden Markov modeling analysis, respectively. The Kramers rate framework yields a considerably low-speed limit of 21 ms for a helical hairpin formation in lipid bilayers, compared to µs scale for soluble protein folding. This large discrepancy is likely due to the highly viscous nature of lipid membranes, retarding the helix-helix interactions. Our results offer a more valid guideline for relating the kinetics and free energies of membrane protein folding.
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Proteínas de la Membrana , Pliegue de Proteína , Proteínas de la Membrana/química , Fenómenos Mecánicos , Cinética , EntropíaRESUMEN
VHH antibodies or nanobodies, which are antigen-binding domains of heavy chain antibodies from camelid species, have several advantageous characteristics, including compact molecular size, high productibility in bacteria and easy engineering for functional improvement. Focusing on these advantages of VHHs, we attempted to establish an immunoassay system for detection of Legionella, the causative pathogen of Legionnaires' disease. A VHH phage display library was constructed using cDNA from B cells of alpacas immunized with Legionella pneumophila serogroup1 (LpSG1). Through biopanning, two specific VHH clones were isolated and used to construct a Legionella detection system based on the latex agglutination assay. After engineering the VHHs and improving the assay system, the sensitive detection system was successfully established for the LpSG1 antigen. The immunoassay developed in this study should be useful in easy and sensitive detection of Legionella, the causative agent of Legionnaires' disease, which is a potentially fatal pneumonia.
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Legionella , Enfermedad de los Legionarios , Anticuerpos de Dominio Único , Humanos , Antígenos , Inmunoensayo , Cadenas Pesadas de InmunoglobulinaRESUMEN
The filamentous cytoskeletal protein microtubule, a polymer of α and ß heterodimers of tubulin, plays major roles in intracellular transport as well as in vitro molecular actuation and transportation. Functionalization of tubulin dimers through covalent linkage facilitates utilization of microtubule in the nanobioengineering. Here we present a detailed description of the methodologies used to modify tubulin dimers with DNA strand and biotin through covalent interaction.
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Biotina , Tubulina (Proteína) , Transporte Biológico , Biotina/metabolismo , ADN/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Small interfering RNAs (siRNA) are attractive and powerful tools to inhibit the expression of a targeted gene. However, their extreme hydrophilicities combined with a negative charge and short plasma half-life counteract their use as therapeutics. Previously, we chemically linked siRNA to squalene (SQ) which self-assembled as nanoparticles (NPs) with pharmacological efficiency in cancers and recently in a hereditary neuropathy. In order to understand the siRNA-SQ NP assembly and fate once intravenously injected, the present study detailed characterization of siRNA-SQ NP structure and its interaction with serum components. From SAXS and SANS analysis, we propose that the siRNA-SQ bioconjugate self-assembled as 11-nm diameter supramolecular assemblies, which are connected one to another to form spherical nanoparticles of around 130-nm diameter. The siRNA-SQ NPs were stable in biological media and interacted with serum components, notably with albumin and LDL. The high specificity of siRNA to decrease or normalize gene expression and the high colloidal stability when encapsulated into squalene nanoparticles offer promising targeted therapy with wide applications for pathologies with gene expression dysregulation.
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Nanopartículas , ARN Interferente Pequeño , Dispersión del Ángulo Pequeño , Escualeno , Difracción de Rayos XRESUMEN
Lipopolyplexes present well-established nucleic acid carriers assembled from sequence-defined cationic lipo-oligomers and DNA or RNA. They can be equipped with additional surface functionality, like shielding and targeting, in a stepwise assembly method using click chemistry. Here, we describe the synthesis of the required compounds, an azide-bearing lipo-oligomer structure and dibenzocyclooctyne (DBCO) click agents as well as the assembly of the compounds with siRNA into a surface-functionalized formulation. Both the lipo-oligomer and the DBCO-equipped shielding and targeting agents are produced by solid-phase synthesis (SPS). This enables for precise variation of all functional units, like variation in the amount of DBCO attachment sites or polyethylene glycol (PEG) length. Special cleavage conditions with only 5% trifluoroacetic acid (TFA) must be applied for the synthesis of the shielding and targeting agents due to acid lability of the DBCO unit. The two-step lipopolyplex assembly technique allows for separate optimization of the core and the shell of the formulation.
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Química Clic , Lípidos/química , Polímeros/química , Fenómenos Biofísicos , Cromatografía en Gel , Polímeros/síntesis química , Polímeros/aislamiento & purificación , Técnicas de Síntesis en Fase SólidaRESUMEN
PNA is hybrid molecule ideally suited for bridging the functional landscape of polypeptides with the structural diversity that can be engineered with DNA nanostructures. However, PNA can be more challenging to work with in aqueous solvents due to its hydrophobic nature. A solution phase method using strain promoted, copper free click chemistry was developed to conjugate the fluorescent dye Cy5 to 2 bifunctional PNA strands as a first step toward building cyclic PNA-polypeptides that can be arranged within 3D DNA nanoscaffolds. A 3D DNA nanocage was designed with binding sites for the 2 fluorescently labeled PNA strands in close proximity to mimic protein active sites. Denaturing polyacrylamide gel electrophoresis (PAGE) is introduced as an efficient method for purifying charged, dye-labeled PNA conjugates from large excesses of unreacted dye and unreacted, neutral PNA. Elution from the gel in water was monitored by fluorescence and found to be more efficient for the more soluble PNA strand. Native PAGE shows that both PNA strands hybridize to their intended binding sites within the DNA nanocage. Förster resonance energy transfer (FRET) with a Cy3 labeled DNA nanocage was used to determine the dissociation temperature of one PNA-Cy5 conjugate to be near 50°C. Steady-state and time resolved fluorescence was used to investigate the dye orientation and interactions within the various complexes. Bifunctional, thermostable PNA molecules are intriguing candidates for controlling the assembly and orientation of peptides within small DNA nanocages for mimicking protein catalytic sites.