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A class of (thio)chromenone derivatives has been identified as suitable ligands for uPAR, a glycoprotein with a prognostic value in a large number of human cancers. The (thio)chromenone agents actively inhibited the binding of uPAR to uPA with a binding affinity of 18.6 nM, reducing cell migration in the wound healing assay by up to 40% without apparent cell motility. The discovery of an uPAR-targeting fluorescent probe was also made in this study that can selectively bind to the membrane uPAR, providing valuable molecular insights into the role of uPAR in cancer metastasis. This study should serve as a basis for the development of new uPAR-targeting agents that can control the metastatic potential of cancer cells with minimal cytotoxicity. [ABSTRACT FROM AUTHOR]

Advanced single-molecule techniques have enabled tracking of nanometer-scale movements of DNA and RNA motor proteins in real time. Previously, we reported an ATP-independent diffusion of transactivation response RNA binding protein (TRBP) on dsRNA, yet the mechanistic details remain elusive. Using single-molecule fluorescence assays, we demonstrate that the diffusion activity of TRBP is coordinated by an independent movement of two subdomains, dsRBD1 and dsRBD2, in which the diffusion distance is determined by the length of a flexible linker domain that connects the two dsRBDs. When the linker is shortened, the diffusion distance is reduced proportionally, suggesting a ruler-like function of the linker domain. Diffusion stalls upon encountering a physical barrier in the form of an RNA:DNA hybrid segment or bulky secondary structures, indicating a dsRNA scanning mode of TRBP. The results display a plausible mechanism of TRBP in scanning for pre-miRNA or pre-siRNA as proper substrates for the RNAi pathway.

The proteins harboring double-stranded RNA binding domains (dsRBDs) play diverse functional roles such as RNA localization, splicing, editing, export, and translation, yet mechanistic basis and functional significance of dsRBDs remain unclear. To unravel this enigma, we investigated transactivation response RNA binding protein (TRBP) consisting of three dsRBDs, which functions in HIV replication, protein kinase R(PKR)-mediated immune response, and RNA silencing. Here we report an ATP-independent diffusion activity of TRBP exclusively on dsRNA in a length-dependent manner. The first two dsRBDs of TRBP are essential for diffusion, whereas the third dsRBD is dispensable. Two homologs of TRBP, PKR activator and R3D1-L, displayed the same diffusion, implying a universality of the diffusion activity among this protein family. Furthermore, a Dicer-TRBP complex on dsRNA exhibited dynamic diffusion, which was correlated with Dicer's catalytic activity. These results implicate the dsRNA-specific diffusion activity of TRBP that contributes to enhancing siRNA and miRNA processing by Dicer.

Correction for '(Thio)chromenone derivatives exhibit anti-metastatic effects through selective inhibition of uPAR in cancer cell lines: discovery of an uPAR-targeting fluorescent probe' by So-Young Chun et al., Chem. Commun., 2025, https://doi.org/10.1039/D4CC05907G. [ABSTRACT FROM AUTHOR]

Photobleaching is an irreversible photochemical process that makes fluorophores not to emit permanently, limiting the observation time of the molecules. Thus, preliminary photobleaching can interfere with most fluorescence spectroscopy and microscopy, especially single‐molecule fluorescence applications. To minimize photobleaching of fluorophores, low excitation power as well as specialized imaging buffers that improve the photostability of fluorophores is highly recommended in single‐molecule fluorescence measurements. Here, we investigated how photobleaching can get affected by the excitation power and buffer components in the single‐molecule buffer system such as Trolox and glucose oxidase/catalase (GOC). We found that the photobleaching rate showed higher‐order photon interaction, indicating that photobleaching rate is decoupled to the amount of fluorescence. We also observed that the photobleaching rate was significantly reduced by fourfold when the combination of Trolox and oxygen scavenging system using either GOC or N2 bubbling was employed in single‐molecule buffer. [ABSTRACT FROM AUTHOR]

Srs2 dismantles presynaptic Rad51 filaments and prevents its re-formation as an anti-recombinase. However, the molecular mechanism by which Srs2 accomplishes these tasks remains unclear. Here we report a single-molecule fluorescence study of the dynamics of Rad51 filament formation and its disruption by Srs2. Rad51 forms filaments on single-stranded DNA by sequential binding of primarily monomers and dimers in a 5'-3' direction. One Rad51 molecule binds to three nucleotides, and six monomers are required to achieve a stable nucleation cluster. Srs2 exhibits ATP-dependent repetitive motion on single-stranded DNA and this activity prevents re-formation of the Rad51 filament. The same activity of Srs2 cannot prevent RecA filament formation, indicating its specificity for Rad51. Srs2's DNA-unwinding activity is greatly suppressed when Rad51 filaments form on duplex DNA. Taken together, our results reveal an exquisite and highly specific mechanism by which Srs2 regulates the Rad51 filament formation.