Bioconjugate Chemistry最新文献

Chemical modifications in RNA therapeutics have addressed major challenges by enhancing metabolic stability, cellular uptake, and biological activity─regardless of their mechanism of action. Here, we report on the synthesis of 4′-C-ethynyl cytidine (4′-C-EthC) and its 2′-O-methylated derivative (4′-C-EthC-2′-OMe) as phosphoramidite building blocks and their subsequent incorporation into oligonucleotides. These ribose C4-terminal alkyne modifications provide a click handle directly within oligonucleotides. The novel modification is accessible via copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) and serves as a universal 4′-C-ribose modifier on the oligonucleotide level. We identified both aromatic and aliphatic triazole residues that increase the thermodynamic stability in A-form RNA duplexes. Furthermore, 4′-C-triazole-modified oligonucleotides exhibit high resistance to nuclease-mediated degradation in metabolic stability assays. Finally, we introduced the novel modification and its substituted triazoles into guide RNAs (gRNAs) for site-directed A-to-I editing in mammalian cells and compared their performance with phosphorothioate-modified gRNAs.

T-cell immunoglobulin and mucin domain 3 (TIM-3), a critical immunosuppressive checkpoint receptor, regulates antitumor immunity within the tumor microenvironment (TME). Noninvasive quantification of TIM-3 expression could be helpful for guiding immunotherapy and monitoring treatment response. We developed ⁶⁸Ga-DOTA-D-P24, a novel D-configured peptide radiotracer designed for enhanced protease resistance and optimized TIM-3 targeting. Radiolabeling yielded high radiochemical purity (RCP) (>98%) and excellent in vitro/in vivo stability. Positron emission tomography (PET)/CT imaging across six tumor models demonstrated that ⁶⁸Ga-DOTA-D-P24 showed high specific uptake in MGC-803 gastric carcinoma. Comparative PET studies showed that the D-configured tracer exhibited 1.6-fold higher tumor uptake than that of ⁶⁸Ga-DOTA-L-P24. Furthermore, ⁶⁸Ga-DOTA-D-P24 successfully visualized the interleukin-15 (IL-15)-triggered elevation of TIM-3 expression in tumors, demonstrating its potential as a noninvasive tool for assessing target engagement and treatment response in TIM-3-associated malignancies.

Targeted delivery of macromolecular therapeutics holds great promise for overcoming the limitations of conventional small molecules, enabling the modulation of protein–protein interactions and precise genome editing. However, efficient, safe, and cell type-specific delivery remains a major challenge. To address this, we developed a modular platform for synthesizing heterotrifunctional bio-orthogonal macromolecular conjugates (BMCs) by engineering diverse combinations of targeting ligands, cell-penetrating peptides (CPPs), and bioactive cargos. We optimized facile bioconjugation chemistries to generate BMCs with improved yields, structural integrity, and activity. Modular BMCs accommodate diverse components, including antibodies and receptor ligands for targeting, CPPs for intracellular trafficking, and optical probes, therapeutic peptidomimetics, and CRISPR-Cas9 nuclease as cargo to confer specific biological activities. We assayed their utility across multiple applications: BMCs with fluorescently labeled cargo revealed endosomal escape and intracellular accumulation; peptidomimetic MYB transcription factor inhibitor BMCs exhibited potent antileukemic activity against acute myeloid leukemia cells; and Cas9 BMCs achieved rapid delivery and cell type-specific gene editing in human cells. The BMC approach enables the customizable delivery of functional macromolecules, nominating BMCs as a broadly applicable platform for biomedical applications.

Glucagon-like peptide-1 receptor (GLP-1R) is overexpressed in >90% of insulinomas, making it an optimal target for imaging. However, current GLP-1R agonist tracers may induce side effects including hypoglycemia and nausea, particularly in pediatric patients. In this study, we employed a rational design approach combining molecular dynamics (MD) simulations with experimental validation to develop three ⁶⁸Ga-labeled NOTA-conjugated exendin(9–39) derivatives featuring antagonist activity for safer imaging. MD simulations predicted differential binding affinities based on conjugation sites at Asp⁰⁹ (E09), Lys¹² (E12), and Lys²⁷ (E27), with MM/GBSA calculations ranking E09 (−216.06 kcal/mol) > E12 (−200.01 kcal/mol) > E27 (−117.08 kcal/mol). Experimental validation through surface plasmon resonance confirmed these computational predictions, showing binding affinities consistent with the computational predictions. All radiotracers achieved radiochemical yields (>95%) and plasma stability (>91% intact after 120 min). In vivo PET imaging validated the computational hierarchy, with [⁶⁸Ga]Ga-E09 demonstrating superior tumor uptake (SUV_max: 3.99 at 60 min) compared with E12 (SUV_max: 0.75 at 60 min) or E27 (undetectable). These findings highlight the power of combining computational screening with systematic experimental validation. In conclusion, [⁶⁸Ga]Ga-E09 demonstrates superior binding affinity, cellular uptake, and imaging performance, suggesting its potential as a promising agent warranting further studies.

Cutaneous melanoma, responsible for 80% of skin cancer mortality, presents urgent diagnostic challenges due to insufficient early detection methods. Current clinical methods rely on invasive biopsies, while noninvasive approaches primarily serve as adjunctive decision-support tools rather than definitive diagnostics. Here, a peptide-based fluorescent biosensing system was developed for the sensitive and rapid detection of S100B, a key prognostic biomarker for melanoma. Our system employs a fluorescently labeled peptide beacon designed for Förster resonance energy transfer (FRET)-based detection, achieving a subnanomolar detection limit (∼0.045 nM) and great selectivity in human serum samples. Peptide synthesis was performed using optimized solid-phase protocols, enabling precise sequence assembly, while the peptide sensor offers efficient detection, lower costs, and high specificity through tailored peptide–protein interactions. The biosensing probe employs complementary peptide nucleic acid (PNA) interactions to achieve proximity-induced fluorescence quenching in the absence of S100B, which reverses via structural rearrangement upon specific S100B binding for accurate quantification. Computational and experimental optimization of the synthetic process has enhanced binding efficiency, sensitivity, and response time–crucial parameters for melanoma-specific detection. By integrating advanced molecular design with optical biosensing, this mechanism aims to enhance the accuracy and accessibility of melanoma diagnostics, ultimately addressing healthcare disparities and improving patient outcomes.

In an attempt to broaden the scope of functional nucleic acids, phosphorescent platinum(II) complexes, resembling artificial metal-containing nucleobases, were attached covalently to DNA oligonucleotides via a deoxyribose moiety. The distance between the deoxyribose and the complex was varied by selecting three different linkers (propylene, ethylene, and methylene). Stable duplexes were obtained with any of the canonical nucleobases in the complementary position. When guanine was placed in this position, the most stable duplexes were obtained. No clear correlation was found between the identity of the linker and duplex stability. When two platinum(II) complexes were placed in adjacent positions within an oligonucleotide strand, photoluminescence spectra exhibited an additional broad low-energy band due to luminescence with excimeric character, indicating Pt···Pt interactions. The ratio of monomeric and excimeric emissions depends on the linker length and, interestingly, on the presence of dioxygen. Hence, a platinated oligonucleotide was developed into a ratiometric dioxygen sensor, capable of rapidly detecting dioxygen levels in volumes as small as 2 μL. The oligonucleotide proved to be nontoxic at relevant concentrations and could be transfected into cells, where it appeared to degrade so that further modification will be necessary to obtain an oligonucleotide-based ratiometric dioxygen sensor for intracellular measurements.

Bicyclic peptides, with two cyclic substructures, have emerged as a powerful tool for modulating challenging targets such as protein–protein interactions. Meanwhile, DNA-encoded library technology (DELT) provides a powerful platform for hit discovery. The unity of both fields has the potential to identify potent bicyclic ligands for the targets of interest. Therefore, there is a high demand to develop an efficient way to construct bicyclic peptide libraries. Herein, we describe a novel and efficient approach to the synthesis of DNA-encoded bicyclic peptides via a cysteine-promoted cyclization and amide condensation reaction. This strategy proceeds smoothly under mild conditions and can generate a wide range of bicyclic peptides with various peptide sequences and ring sizes in good conversions.

Messenger RNA (mRNA) lipid nanoparticles (LNPs) have emerged as a transformative technology with broad applications in vaccines, protein replacement therapy, and gene editing. However, the transient nature of mRNA expression often necessitates high or repeated dosing regimens, limiting its therapeutic potential. Thus, there is a critical need for innovation at the interface of RNA biology and drug delivery that prolong the duration of RNA translation. In this Viewpoint, we provide an overview of emerging nucleic acid cargos that address these challenges, specifically self-amplifying RNA (saRNA) and circular RNA (circRNA), and provide a framework for how these nucleic acid cargos can enable the next generation of vaccines and therapeutics for diverse clinical applications.

The effect of polymer bioconjugation on the binding properties of the ATP aptamer was studied using bio-layer interferometry (BLI) and surface plasmon resonance (SPR). For this study, ten different polymer-aptamer bio-hybrids were synthesized by automated solid-phase phosphoramidite chemistry. These bio-hybrid macromolecules contain (i) a DNA segment (i.e., either the ATP aptamer or a control sequence with no ATP affinity), (ii) one or two synthetic poly(phosphodiester) segments containing either propyl, triethylene glycol, or pentaethylene glycol spacers, and (iii) a biotin end-group allowing immobilization on streptavidin sensors for BLI and SPR experiments. Diblock and triblock architectures were prepared in order to assess the influence of the number of conjugated polymer chains on aptamer-target binding. All bio-hybrid polymers were characterized by high resolution electrospray mass spectrometry, ion-exchange HPLC, and polyacrylamide gel electrophoresis. All these methods confirmed the formation of the targeted bio-hybrids. Furthermore, BLI and SPR experiments demonstrated that all bio-hybrid macromolecules containing the ATP aptamer sequence could bind ATP, indicating that polymer conjugation did not compromise the aptamer’s functionality, even when positioned between two synthetic chains.

High glutathione levels in the tumor microenvironment drive tumor resistance and proliferation, making glutathione depletion a key cancer therapeutic strategy. Existing inhibitors face issues such as poor specificity and biocompatibility, creating a need for new formulations with high targeting, low toxicity, and efficient depletion. This study synthesized carbon dots (PCDs) via the hydrothermal method using 3,4,9,10-perylenetetracarboxylic dianhydride as a precursor. PCDs oxidize glutathione for depletion via intrinsic redox properties and act as efficient glutathione probes with fluorescence and colorimetric detection limits of 0.527 and 13.11 μM, respectively, showing excellent stability in complex biological environments. Under an 808 nm near-infrared laser, PCDs exhibit 41.88% photothermal conversion efficiency. PCDs induce tumor cell apoptosis by depleting glutathione with enhanced antitumor effects under photothermal therapy synergy. They disrupt tumor redox homeostasis to trigger immunogenic cell death, promote dendritic cell maturation, polarize M2 macrophages to M1, and activate T cell-mediated immunity. In vivo dual-tumor models confirmed that PCDs combined with αPD-L1 efficiently ablate primary tumors, inhibit distal growth, and exert systemic antitumor immune effects. This simple-synthesized, biocompatible PCDs integrate detection and antitumor functions, offering new ideas for next-generation nanodiagnostic/therapeutic materials and combination therapy.