Filter-Assisted ICP-MS Tumor Liquid Biopsy Enabled by Dual-Target-Regulated Functional DNA Nanospheres Cascade AmplificationWu, Wang, Yan
et alSmall Methods (2025)
Abstract: An ultrasensitive ICP-MS aptasensor is developed utilizing a label-free, simple filter membrane-assisted separation technique combined with nucleic acid signal amplification for the analysis of circulating tumor cells (CTCs) in lung cancer clinical samples. The approach is based on the high-affinity interaction between aptamers and PD-L1 and mucin 1, which are overexpressed on the cell surface, in conjunction with functional Y-DNA nanospheres and catalytic hairpin assembly amplifications, enabling the simultaneous detection of two proteins. Additionally, a four-armed nanostructure with significant spatial site resistance is self-assembled by introducing streptavidin with biotinylated-hairpin structures, improving the separation efficiency of the filter membrane. This structural design enables the effective isolation of biotin-T-Hg2+-T and biotin-C-Ag+-C from free Hg2+ and Ag+, facilitating highly sensitive dual-protein detection via ICP-MS. The limits of detection reached ag mL-1 levels for proteins and single-cell levels for A549 cells. CTCs are extracted from whole blood samples of lung cancer patients within 45 min through a simple centrifugation procedure. Quantification of CTCs is performed in 37 clinical samples, demonstrating results consistent with clinical diagnoses. The assay exhibits a specificity of 100% and a sensitivity of 94.5%.© 2025 Wiley‐VCH GmbH.
CDRxAbs: Antibody Small-Molecule Conjugates with Computationally Designed Target-Binding SynergyWang, Aceves, Friesenhahn
et alProtein Eng Des Sel (2025)
Abstract: Bioconjugates as therapeutic modalities combine the advantages and offset the disadvantages of their constituent parts to achieve a refined spectrum of action. We combine the concept of bioconjugation with the full atomic simulation capability of computational protein design to define a new class of molecular recognition agents: CDR-extended antibodies, abbreviated as CDRxAbs. A CDRxAb incorporates a covalently attached small molecule into an antibody/target binding interface using computational protein design to create an antibody small-molecule conjugate that binds tighter to the target of the small molecule than the small molecule would alone. CDRxAbs are also expected to increase the target binding specificity of their associated small molecules. In a proof-of-concept study using monomeric streptavidin/biotin pairs at either a nanomolar or micromolar-level initial affinity, we designed nanobody-biotin conjugates that exhibited >20-fold affinity improvement against their protein targets with step-wise optimization of binding kinetics and overall protein stability. The workflow explored through this process promises a novel approach to optimize small-molecule based therapeutics and to explore new chemical and target space for molecular-recognition agents in general.© The Author(s) 2025. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Ultrasensitive determination of exosomes by tyramine-assisted colorimetric sensors for tumor diagnosisKong, Li, Li
et alAnalyst (2025)
Abstract: Exosomes, which are recognized as a kind of valuable liquid biopsy biomarker, exhibit significant application potential in cancer diagnosis. Therefore, it is crucial to establish a reliable detection method for their clinical application. In this study, we have presented an ultrasensitive aptasensor for the visual detection of exosomes by employing tyramine-assisted dual-signal amplification technology. First, we utilized magnetic beads modified with the nucleolin aptamer (MNPs-Aptnucleolin) to capture exosomes. This modification not only enhanced specificity, but also reduced interference of complex sample components. The captured exosomes as a rich source of proteins can bind with multiple biotinyl-tyramide (Bio-TR) molecules through a catalytic reaction involving horseradish peroxidase (HRP) and H2O2. Second, streptavidin-HRP complex-modified gold nanoparticles (GNPs-Str-HRP) as a signal amplification probe was introduced to further enhance the detection signal by binding to Bio-TR. Lastly, the addition of 3,3',5,5'-tetramethylbenzidine (TMB) solution induced a visible color change, enabling quantification of the exosome concentration. This dual-signal amplification strategy resulted in a low limit of detection (LOD) of 63 particles per μL, and it also demonstrated accurate visual diagnosis capabilities for clinical samples. The successful implementation of this approach suggests its potential as a promising tool for point-of-care testing (POCT) in cancer diagnostics.
Application of a novel RNA-protein interaction assay to develop inhibitors blocking RNA-binding of the HuR proteinFilcenkova, Reisbitzer, Joseph
et alFront Genet (2025) 16, 1549304
Abstract: RNA-protein interactions play an important regulatory role in several biological processes. For example, the RNA-binding protein HuR (human antigen R) binds to its target mRNAs and regulates their translation, stability, and subcellular localization. HuR is involved in the pathogenic processes of various diseases. Thus, small molecules blocking RNA-binding of HuR may be useful in a variety of diseases. Previously, we identified STK018404 as a small molecule targeting the HuR-RNA interaction. Based on this study we identified optimized compounds by exploiting combined structure-based and ligand-based computational approaches. To test a series of these compounds, we developed a novel readout system for the HuR-RNA interaction. Traditional methods to detect RNA-protein interaction come with some disadvantages: they require significant reagent optimization and may be difficult to optimize for weakly expressed RNA molecules. The readout often requires amplification. Thus, these methods are not well suited for quantitative analysis of RNA-protein interactions. To achieve an easy-to-perform, rapid, and robust detection of RNA-protein binding, we applied a split luciferase reporter system, to detect the interaction between HuR and its target RNA. We expressed one luciferase fragment as a fusion protein with HuR. The second luciferase fragment was Streptavidin-coated and coupled to a biotinylated RNA-oligo comprising an AU-rich HuR-binding element. The binding between HuR and its target RNA-oligo then allowed reconstitution of the functional luciferase that was detectable by luminescence. Using the split luciferase reporter system, we present here a series of optimized compounds that we developed.Copyright © 2025 Filcenkova, Reisbitzer, Joseph, Weber, Carloni, Rossetti and Krauß.
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