Xue Qiu

To address the emerging need for high-throughput and sensitive methodologies in RNA-targeted drug discovery, this study developed and validated a novel biosensing platform based on time-gated Förster resonance energy transfer (TG-FRET). The highly conserved RNA pseudoknot of SARS-CoV-2 served as our model system and target, a structure that regulates the essential viral –1 programmed ribosomal frameshift (−1 PRF). We first constructed a fluorescent protein (mOrange2-mCherry)-based FRET reporter system to visualize and monitor –1 PRF efficiency in living cells. To enable high-throughput screening, we adapted the system into a TG-FRET assay. This assay was based on a HaloTag-mCherry fusion protein, in which the HaloTag moiety was site-specifically labeled with CoraFluor, a new class of macrotricyclic terbium complexes. This configuration enables homogeneous, time-gated detection directly in cell lysates, effectively eliminating interference from biomatrix autofluorescence. Using this platform, we quantified ivacaftor’s inhibition of −1 PRF (IC50 = 7.6 μM) and ruled out direct inhibition by MTDB. The established TG-FRET platform combines the precision for detecting molecular interactions with the high-throughput capability compatible with microplate operations, providing a powerful and versatile screening tool for drug discovery targeting functional RNA structures.

Discovering new E3 ligase ligands and accurate characterizations of the critical binary interactions between these ligands and the ligases are fundamental to the rational design of new E3 ligase ligands and the expansion of proteolysis-targeting chimeras. Herein, we developed a time-resolved Förster resonance energy transfer (TR-FRET) platform for multiparameter profiling of protein-ligand interactions to access previously obscured insights from conventional affinity-based assays. This platform was employed to investigate interactions ranging from model systems (HaloTag and VHL) to novel E3 ligase CDC20 with Apcin. From the multiexponential fitting of photoluminescence decays, key parameters (distinct FRET subpopulations, their amplitudes, efficiencies, and distances) directly report the protein binding site occupancy (or fraction of bound ligand) as well as potential alterations in binding geometry or molecular orientation. The heatmap visualization further converts complex binding data into an intuitive format, providing medicinal chemists with a practical and expandable tool to guide ligand optimization across diverse target proteins and E3 ligases.

The development of glycoscience has been impeded by the inherent structural complexity of carbohydrates, the lack of template encoding, and the limitations of current methodologies for their identification and quantification. Carbohydrate-binding modules (CBMs) and carbohydrate-active enzymes (CAZymes) are two protein families that have evolved to interact with distinct carbohydrates, offering the potential to function as specific carbohydrate recognition elements. Here, we present the first homogeneous assays for alginate as a representative polysaccharide, leveraging the unique properties of CBMs and CAZymes (e.g., polysaccharide lyases). In contrast to conventional antibody-antigen-antibody "sandwich" immunoassays, our approach takes advantage of the linear and repetitive nature of polysaccharides, allowing for simultaneous binding to multiple CBMs and lyases. We exploit this feature in our novel "Barbecue" assay design, utilizing the combination of CBMs/lyases that are orthogonally labeled with the Tb-based donor fluorophore CoraFluor-1 (CRF) and Cy5 as an acceptor to establish a time-resolved Förster resonance energy transfer (TR-FRET) detection system. The homogeneous single-step assay uses a straightforward protocol without wash steps, enabling the sensitive quantification of alginate. The lower limit of quantification was determined to be as low as 0.4 ng·mL^-1 (0.01 ng) with CBMs and 1.6 ng·mL^-1 (0.03 ng) with alginate lyases, representing a ≥100-fold improvement compared to that of conventional microarray-based assays. The broad applicability of the analytical method was rigorously validated across biomedical, food, and cosmetics samples. The "Barbecue" FRET concept is generalizable and can be applied to develop similar rapid and simple quantification assays for other polysaccharides beyond alginate.

Accurate quantification of site-specific tau phosphorylation in plasma holds great promise for the noninvasive early diagnosis of Alzheimer's disease (AD). Here, we integrated the proximity extension assay (PEA) with nucleic acid amplification techniques-polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA)-and coupled them with CRISPR/Cas12a-mediated fluorescence detection to enable quantitative and homogeneous measurement of threonine-181-phosphorylated tau (p-tau181), a key biomarker of AD. Binding of two PEA probes to a single p-tau181 molecule induces proximity-mediated probe hybridization and extension, thereby converting the protein signal into an amplifiable nucleic acid signal. The resulting double-stranded DNA is subsequently amplified by PCR or RPA and detected through Cas12a trans-cleavage activity. The limits of detection (LODs) for the PEA-PCR-CRISPR/Cas and PEA-RPA-CRISPR/Cas assays were 149.0 fM (6.8 pg·mL^-1) and 45.4 fM (2.1 pg·mL^-1), respectively. In fetal bovine serum, LODs of 231.4 fM (10.6 pg·mL^-1) and 139.2 fM (6.3 pg·mL^-1) were achieved, demonstrating excellent antimatrix performance. The accuracy of the PEA-RPA-CRISPR/Cas assay in human serum was further validated using a commercial enzyme-linked immunosorbent assay (ELISA) kit. This homogeneous, wash-free approach combines operational simplicity with ultrahigh sensitivity, showing great potential for routine clinical detection and early stage monitoring of AD biomarkers.

Glutathione peroxidase 4 (GPX4) plays a crucial role in the ferroptosis pathway, emerging as a potential drug target in the treatment of refractory tumors.

Aberrant FGF2/FGFR signaling is implicated in lung squamous cell carcinoma (LSCC), posing treatment challenges due to the lack of targeted therapeutic options. Designing drugs that block FGF2 signaling presents a promising strategy different from traditional kinase inhibitors. We previously reported a ColVα1-derived fragment, HEPV (127AA), that inhibits FGF2-induced angiogenesis. However, its large size may limit therapeutic application. This study combines rational peptide design, molecular dynamics simulations, knowledge-based prediction, and GUV and FRET assays to identify smaller peptides with FGF2-blocking properties. We synthesized two novel peptides, HBS-P1 (45AA) and HBS-P2 (66AA), that retained the heparin-binding site. Both peptides demonstrated anti-LSCC and antiangiogenesis properties in cell viability and microvessel network induction assays. In two LSCC subcutaneous models, HBS-P1, with its affinity for FGF2 and enhanced penetration ability, demonstrated substantial therapeutic potential without apparent toxicities. Our study provides the first evidence supporting the development of collagen V-derived natural peptides as FGF2-blocking agents for LSCC treatment.

Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an “APPROACH” strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ng∙μL⁻¹, 0.77 ng∙μL⁻¹, and 1.1 ng∙μL⁻¹, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ng∙μL⁻¹ in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy.

Alpha-synuclein (α-Syn) is a key protein of Parkinson's disease (PD). Oligomers formed by misfolding and aggregation of α-Syn can cause many pathological phenomena and aggravate the development of PD. Therefore, sensitive and accurate detection of oligomers is essential to understanding the pathology of PD and beneficial to screening and developing new drugs against PD. Here, we demonstrated a simple and sensitive method to detect the early aggregation of α-Syn via Förster resonance energy transfer (FRET) technology. We performed systematic investigations of the FRET sensitizations, efficiencies, and donor-to-acceptor distances during α-Syn aggregation, which was proved to be more sensitive to reflect small distance changes in the early stage of α-Syn aggregation, especially for α-Syn oligomers. The FRET assays were also applied to study the influence of Ser129 phosphorylation (pS129) on the aggregation rate of α-Syn. Our results showed that pS129 modification promotes α-Syn aggregation and enhances the ability of preformed fibrils to induce monomer aggregation. pS129 also increased the cytotoxicity of α-Syn. These results are of great significance for a better understanding of the pathological mechanisms of PD and future PD drug development.