Bioconjugate Chemistry最新文献

G-quadruplex (G4) DNA structures are noncanonical secondary structures found in key regulatory regions of the genome, including oncogenic promoters and telomeres. Small molecules, known as G4 ligands, capable of stabilizing G4s hold promise as chemical probes and therapeutic agents. Nevertheless, achieving precise specificity for individual G4 structures within the human genome remains a significant challenge. To address this, we expand upon G4-ligand-conjugated oligonucleotides (GL-Os), a modular platform combining the stabilizing properties of G4-ligands with the sequence specificity of guide DNA oligonucleotides. Central to this strategy is the linker that bridges the G4 ligand and the guide oligonucleotide. In this study, we develop multiple conjugation strategies for the GL-Os that enabled a systematic investigation of the linker in both chemical composition and length, enabling a thorough assessment of their impact on targeting oncogenic G4 DNA. Biophysical, biochemical, and computational evaluations revealed GL-Os with optimized linkers that exhibited enhanced binding to target G4s, even under thermal or structural stress. Notably, longer linkers broadened the range of targetable sequences without introducing steric hindrance, thereby enhancing the platform's applicability across diverse genomic contexts. These findings establish GL-Os as a robust and versatile tool for the selective targeting of individual G4s. By facilitating precise investigations of G4 biology, this work provides a foundation for advancing G4-targeted therapeutic strategies and exploring their role in disease contexts.

G-quadruplex (G4) DNA structures are noncanonical secondary structures found in key regulatory regions of the genome, including oncogenic promoters and telomeres. Small molecules, known as G4 ligands, capable of stabilizing G4s hold promise as chemical probes and therapeutic agents. Nevertheless, achieving precise specificity for individual G4 structures within the human genome remains a significant challenge. To address this, we expand upon G4-ligand-conjugated oligonucleotides (GL-Os), a modular platform combining the stabilizing properties of G4-ligands with the sequence specificity of guide DNA oligonucleotides. Central to this strategy is the linker that bridges the G4 ligand and the guide oligonucleotide. In this study, we develop multiple conjugation strategies for the GL-Os that enabled a systematic investigation of the linker in both chemical composition and length, enabling a thorough assessment of their impact on targeting oncogenic G4 DNA. Biophysical, biochemical, and computational evaluations revealed GL-Os with optimized linkers that exhibited enhanced binding to target G4s, even under thermal or structural stress. Notably, longer linkers broadened the range of targetable sequences without introducing steric hindrance, thereby enhancing the platform’s applicability across diverse genomic contexts. These findings establish GL-Os as a robust and versatile tool for the selective targeting of individual G4s. By facilitating precise investigations of G4 biology, this work provides a foundation for advancing G4-targeted therapeutic strategies and exploring their role in disease contexts.

We report the chemical conjugation of a recombinant Adeno Associated Virus (rAAV) capsid with various functionalities, including proteins, using a bioorthogonal strategy. rAAVs were azido-coated or dibenzylcyclooctyne (DBCO)-coated by chemically modifying lysine or tyrosine residues. Lysine residues were modified using a phenyl isothiocyanate anchor, and tyrosine residues using either an aryl diazonium salt or a N-methyl luminol derivative. We demonstrate anchor-dependent labeling levels, as observed with biochemical assays and mass spectrometry. Strain-promoted azide-alkyne cycloaddition (SPAAC) was then implemented and evaluated on the rAAV to append functionalities such as fluorescein, biotin, and carbohydrates to the azido-coated capsids. We confirmed the efficiency of the bioorthogonal reaction and observed a stronger reactivity with dibenzylcyclooctyne (DBCO) compared to bicyclononyne (BCN). The optimized SPAAC reaction was finally used to label the viral vectors with two relevant nanobodies targeting specific immune cell receptors (CD62L and CD45). In vitro transduction assays conducted with one rAAV-nanobody conjugate demonstrated the promising targeting properties of these chemically modified vectors. Thus, we anticipate that this strategy will positively impact the field of rAAV capsid engineering and contribute in tissue-specific targeting for the optimization of gene therapy treatments.

This study explores the use of lateral flow assays (LFAs), recognized for their simplicity and ease-of-use, as a tool for characterizing nanoparticles functionalized with various biomolecules (e.g., proteins, antibodies, and nucleic acids). A half-strip model system was developed using ovalbumin (OVA) conjugated to gold nanoparticles (AuNPs). The characterization results obtained with LFAs were compared to those from traditional methods such as infrared spectroscopy and fluorescence labeling. The advantages of LFAs in characterizing such conjugated nanosystems were clearly demonstrated. The use of half-strip assays could not only confirm the presence of OVA on AuNPs but also enable the quantification of OVA bound per nanoparticle, offering a rapid and quantitative characterization method. Additionally, the assay showcased its versatility, as it was successfully applied to optimize the covalent coupling conditions of OVA on AuNPs, as well as to differentiate between covalently bound and adsorbed proteins. Furthermore, LFAs were employed to detect antibodies on functionalized nanoparticles, optimize their coupling to a newly developed organic coating, and confirm both the grafting of nucleic acids onto the surface and their pairing with complementary strands. These findings underscore the remarkable adaptability of LFAs for characterizing diverse nanoconjugates. Overall, LFAs stand out as a versatile and accessible tool for characterizing complex bioconjugated nanosystems, making them highly suitable for rapid Quality Control (QC) analysis and bioconjugation optimization.

Heat-induced cancer therapies such as magnetic hyperthermia-based cancer therapy (MHCT) and photothermal tumor ablation (PTT) have garnered significant attention as minimally invasive new-generation cancer therapy modalities. However, solid tumors associated with hypoxia present a considerable challenge to effective cancer therapy. In this study, we took up the challenge of mitigating the limiting penetration ability of nanoparticles by integrating polydopamine-coated magnetic nanoparticles and motile anaerobic bacteria (PDBs) to function as a smart thermo-vector. The developed PDBs are capable of self-navigating hypoxic tumors and as thermo-therapy agents with the ability to induce heat through exposure to an alternating magnetic field or near-infrared laser light. The thermo-vector system exhibited a dual-functioning synergistic antitumor effect of MHCT and PTT and an outstanding tumor targeting efficiency, outperforming the conventional 'nanoparticles only' approach. The heat-induced cellular oxidative stress and disrupted mitochondrial function led to 80% cellular cytotoxicity within 24 h of treatment. The PDB-based approach led to complete tumor regression in c57BL/6 mice within 21 days of treatment and a tumor-free survival for 60 days without recurrence, proving the capability of the developed PDBs in combatting solid tumors.

In order to effectively replicate within a host cell, the Legionella pneumophila bacterium secretes effector enzymes into the cytoplasm in order to manipulate cellular host pathways including host ubiquitination. Some of these effectors, the so-called SidE-family, mediate noncanonical phosphoribosyl serine ubiquitination (PR-ubiquitination) of host substrate proteins, contributing to the recruitment of ER-remodeling proteins and the formation of a Legionella-containing vacuole, which is crucial in the early stages of bacterial infection. PR-ubiquitination is a dynamic process that is reversed by other Legionella effectors called deubiquitinases for PR-ubiquitination (Dups). We recently discovered a reactive allosteric cysteine in close proximity to the catalytic triad of DupA, which can be exploited as a target for covalent probe development. We here report on the synthesis of vinyl-sulfonate and fluoro-sulfonate warhead-containing phosphoribosyl ubiquitin probes, where the Arg42 position of ubiquitin is linked to the C1 of ribose via a native guanidinium group, and compare them to triazole-linked probes. In vitro tests on recombinant DupA and SdeA_PDE revealed that these probes are able to capture the enzymes covalently. In a pull-down proteomics experiment, DupA and DupB enzymes are enriched from Legionella-infected cell lysates, highlighting the potential of native Arg-riboside linked probes to capture Legionella effector enzymes in a complex proteome.

Lysosome-targeting chimeras (LYTACs) harness the cell's lysosomal degradation machinery to break down extracellular and membrane proteins. Previous methods used a synthetic glycopeptide containing multiple serine-O-mannose-6-phosphate (poly-M6Pn), which presented challenges such as synthetic complexity and potential immunogenicity associated with poly-M6Pn. This study introduced a LYTAC formulation, LYTAC^gyM6pG, which uses glyco-engineered yeast-derived mannose-6-phosphate glycans (gyM6pGs) for lysosomal transport, overcoming synthetic complexities and immunogenic risks. The gyM6pGs used in LYTAC^gyM6pG are human-compatible (identical to the structures found in humans) and are efficiently produced through yeast fermentation, followed by the preparation of cell wall glycans and their in vitro modifications. We employed copper-free click chemistry (azide and dibenzocyclooctyne reactions) for the robust conjugation of gyM6pGs to a nanobody targeting the immune checkpoint protein PD-L1, thereby streamlining the assembly of LYTAC^gyM6pG. We demonstrated that LYTAC^gyM6pG effectively degraded endogenous and recombinant PD-L1 proteins on the cell surface by directing them to the lysosome via the cation-independent mannose-6-phosphate receptor pathway. Furthermore, LYTAC^gyM6pG significantly enhanced T cell-mediated cytotoxicity against cancer cells, surpassing the efficacy of nanobodies alone. The successful application of gyM6pGs in the development of LYTAC^gyM6pG highlights the potential for a more viable and scalable therapeutic production of LYTACs, paving the way for broader therapeutic applications, including cancer treatment.

Post-translational modifications (PTMs) on histones play a crucial role in determining the structure and function of chromatin, thereby regulating the eukaryotic gene expression. Histone lysine methylation and acetylation are among the most widespread and biomedically important PTMs, with new chemical tools for their examination in high demand. Here, we report the first use of γ-selenalysine as an efficient lysine mimic for enzymatic methylation, acetylation, and deacetylation reactions catalyzed by histone lysine methyltransferases, acetyltransferases, and a deacetylase. We also show that easily accessible selenocysteine and cysteine residues can undergo chemo- and site-selective alkylation reactions to generate both unmodified and modified γ-selenalysine and related γ-thialysine residues in histone peptides. This dual-modification strategy enables the site-specific incorporation of two distinct functionalities into peptides, mimicking lysine post-translational modifications commonly found on histones. Our research presents a novel approach in which selenocysteine serves as a unique handle for the chemoselective introduction of selenalysine, along with its methylated and acetylated analogues. These tools are designed to facilitate the study of epigenetic proteins that are important for human health and disease.

Geranylgeranylation is a critical post-translational modification essential for various cellular functions. However, current methods for synthesizing geranylgeranylated proteins are complex and costly, which hinders access to these proteins for both biophysical and biomaterials applications. Here, we present a method for the one-pot production of geranylgeranylated proteins in Escherichia coli. We engineered E. coli to express geranylgeranyl pyrophosphate synthase (GGS), an enzyme that catalyzes the production of geranylgeranyl pyrophosphate. By coexpressing GGS with a geranylgeranyltransferase, we achieved efficient geranylgeranylation of model protein substrates, including intrinsically disordered elastin-like polypeptides (ELPs) and globular proteins such as mCherry and the small GTPases RhoA and Rap1B. We examined the biophysical behavior of the resulting geranylgeranylated proteins and observed that this modification affects the phase-separation and nanoassembly of ELPs and lipid bilayer engagement of mCherry. Taken together, our method offers a scalable, versatile, and cost-effective strategy for producing geranylgeranylated proteins, paving the way for advances in biochemical research, therapeutic development, and biomaterial engineering.

Bacterial keratitis is a prevalent, and severe corneal illness resulting from bacterial pathogens. Failure to administer a timely and suitable therapy may lead to corneal opacity, ulceration, significant vision impairment, or potential blindness. Current clinical interventions for bacterial keratitis involve the administration of topical antimicrobial agents and systemic antibiotics. However, the misuse and overuse of antibiotics have led to the rapid emergence of antibiotic-resistant bacteria. Additionally, the restricted antibacterial spectrum and possible adverse effects of antibiotics have provided considerable obstacles to traditional therapies. This highlights the urgent need for novel and highly effective antimicrobial agents. Antimicrobial peptides (AMPs) are a class of naturally occurring or synthetically designed small molecules that have gained significant attention due to their unique antimicrobial mechanisms and low risk of resistance development. AMPs exhibit promising potential in treating bacterial keratitis through direct antibacterial mechanisms, such as inhibiting cell wall synthesis, disrupting cell membranes, and interfering with nucleic acid metabolism, as well as indirect mechanisms, including modulation of the host immune response. This review provides a comprehensive overview of the antibacterial mechanisms of AMPs and their advancements in the treatment of bacterial keratitis. It emphasizes the role of various modification strategies and artificial-intelligence-assisted design in enhancing the antibacterial efficacy, stability, and biocompatibility of AMPs. Furthermore, this review discusses the latest progress in combining AMPs with delivery systems for improved therapeutic outcomes. Finally, the review highlights the current challenges and future perspectives of AMPs in bacterial keratitis treatment, providing valuable insights for developing novel AMPs with high antibacterial efficacy, stability, and safety for bacterial keratitis therapies.