In Illumina next-generation sequencing, target DNA fragments are ligated to adapters containing chemically modified uracil bases, which are subsequently cleaved by uracil-specific excision reagent (USER) to generate the Y-shaped structure essential for downstream sequencing. Herein, we employed the trans-cleaving F-8 DNAzyme as a starting scaffold (DNAzyme reagent) and performed site-directed mutagenesis within its catalytic core. By combining highly active mutations, we identified the variant F-85A6A, which exhibited the highest cleavage yield and demonstrated superior adapter-cleavage efficiency relative to USER, underscoring its potential as a cost-effective USER substitute. We then integrated the catalytic core of the F-8 DNAzyme into the Illumina adapter architecture to design F-8H1. Sequential optimization of the binding arm length and further mutagenesis yielded the highly active mutant H6-13A42A. The 5′ end of H6-13A42A was modified with a phosphate group to construct a complete self-cleaving adapter. In a simulated dual-adapter sequencing structure, the self-cleaving adapter achieved cleavage efficiency comparable to that of USER-treated Illumina adapters while reducing reagent costs by 97.5%, thereby offering an economical molecular tool for advancing DNA sequencing technology.
Existing chemical phosphorylation methods have remained inherently one-way processes, because phosphoryl-group installation either transforms the phosphorylating reagent by changing its oxidation state or consumes hydrolytically labile reactive species, thereby precluding regeneration of the active phosphorylating reagent. Herein, we report the chemical (de)phosphorylation platform in which phosphorylation of histidine and heteroatom nucleophiles is reversed, regenerating both the substrate and the phosphorylating reagent. This switchability further enables phosphoryl-group transfer or rephosphorylation reactions.
Despite recent advances in the synthesis and structural characterization of carbon-centered gold(I) clusters, the fundamental nature of their C−Au bonding and its relationship with the number of Au atoms remain elusive. Here, we report a comprehensive theoretical investigation of CAuIn (n = 6, 5, 4) clusters protected by N-heterocyclic carbene and triphenylphosphine ligands, revealing a systematic evolution of the bonding architecture. Using adaptive natural density partitioning analysis, we identified the transition from classical two-center two-electron (2c−2e) C−Au bonds in the CAu4 core to delocalized three-center two-electron (3c−2e) Au−C−Au bonds specific to the CAu6 core. Notably, the CAuI5 cluster acts as a structural bridge, featuring two axial 2c−2e bonds with orbital characteristics resembling those of the 3c−2e bonds in CAuI6, together with three equatorial 2c−2e bonding environments. This study demonstrates that the relative contributions of the central carbon 2s and 2p orbitals serve as a key electronic descriptor of the nature of C−Au bonding. Furthermore, the coordinated ligands influence the structural distortion of the CAu6 core. Smaller ligands tend to favor symmetric CAu6 cores, whereas bulkier ligands are associated with greater core distortion and a preference for asymmetric CAu6 cores. Both the distortion of the CAu6 core and the chiral ligands contribute to the chirality of the entire metal cluster. This study provides an integrated bonding model that correlates the number of Au atoms in the cluster, electronic structure, ligand effects, stability, and chirality, offering valuable insights for the rational design of functionalized gold(I) nanoclusters.
Abstract Mechanochemical activation represents a powerful concept to harness mechanical force and drive chemical transformations in a solid state. Herein, we report a framework to determine the effect of temperature increase on mechanochemical ball milling in Rh(I)-catalyzed C−H/C−N activation. By using collision dynamics, we provide a model to determine the effect of ball size, milling frequency, internal jar length, velocity, material, and internal curvature during the collisions. By conducting computational studies, we investigate the reaction mechanism and the role of thermal mechanochemical collisions on C−H/C−N bond activation steps. Taking the solution-phase mechanism as a working hypothesis, the observed ball-milling acceleration is consistent with a proposal combining local temperature elevation at impact sites, estimated at up to 83.5 K above the bulk temperature, and modest, step-dependent mechanical modulation of selected transition states, while the rate-determining step is not significantly affected by compression. These results suggest that mechanical force can lower selected transition-state barriers at hot spots, offering a promising strategy for mechanochemical synthesis.
The RNA-binding protein HuR (ELAVL1) is frequently overexpressed and aberrantly localized in cancer cells, where it enhances the stability and translation of numerous transcripts involved in proliferation, survival, invasion, and resistance to therapy. HuR contains multiple RNA-recognition motifs and regulates a broad network of oncogenic mRNAs. Consequently, inhibition of a single RNA-binding pocket is often insufficient to fully suppress its oncogenic activity, as HuR may retain RNA-binding and regulatory functions through its remaining domains. Complete elimination of HuR and its associated ribonucleoprotein complexes may provide a more effective therapeutic strategy than conventional occupancy-based inhibition. Here, we present an induced-proximity approach that redirects HuR-containing complexes toward lysosomal degradation. We developed bifunctional molecules designed to simultaneously bind HuR and engage p62, a selective autophagy receptor that promotes cargo sequestration and subsequent lysosomal clearance. The lead compound induced efficient HuR degradation in breast cancer cells through a lysosome-dependent mechanism. This degradation was accompanied by reduced expression of major HuR-regulated oncogenic targets and a marked decrease in breast cancer cell proliferation and viability. These findings establish p62-mediated lysosomal degradation of HuR complexes as a promising anticancer strategy and provide a broader framework for targeting oncogenic RNA-binding protein networks. More broadly, this work provides a foundation for a next-generation therapeutic platform aimed at targeting previously difficult-to-drug oncogenic RNA-protein networks.
