Specific and sensitive DNA hybridization plays a key role in biotechnology, nanotechnology, and medical technology. However, traditional DNA hybridization-based strategies often require careful tuning of the binding affinity of the probe to attain a trade-off between specificity and sensitivity. Herein, we proposed energy barrier-gated dynamic selectivity to overcome this limitation. The mismatch closure-mediated strand displacement reaction (mcSDR) induces structural constraints through helper strand binding at mismatched sites, resulting in the displacement of the mismatch target requires overcoming an additional activation energy barrier, whereas the perfect match target proceeds via a normal pathway. The mcSDR has been thermodynamically and kinetically demonstrated to be able to balance specificity and sensitivity simultaneously. The energy barrier height can be programmably adjusted by design of helper strand and works in synergy with the toehold exchange strategy to achieve multi-parameter optimization. The superior properties of the mcSDR facilitated the identification of 12 mutation types exhibits excellent specificity in 28 clinically relevant single nucleotide variations. By combining polymerase chain reaction, mutations with an abundance of 0.1% were successfully detected in plasmid samples, and a triple mcSDR was successfully constructed. Clinical validation of 95 glioma and 93 colorectal cancer samples showed that IDH1 and KRAS mutations were 100% consistent with Sanger sequencing. The energy barrier-driven identification mechanism and operational simplicity of mcSDR make it promising for wide applications in biomedical research, molecular diagnosis, and precision medicine.

Platelet storage lesion (PSL) severely impairs platelet function during storage, presenting a major hurdle in transfusion medicine; however, the dynamic interplay between global proteomic changes and post-translational modifications (PTMs) underlying these functional deteriorations remains insufficiently characterized. Here, we report the first comprehensive multi-omics analysis integrating global proteomics, acetylomics, and succinylomics to dissect the molecular dynamics during platelet storage. We performed quantification of global proteomics, acetylome and succinylome based on TMT-labeled LC-MS/MS analysis, combined with antibody-affinity enrichment and purification. Dynamic molecular changes and functional transformation of platelet were also characterized under proper conditions stored for 1, 3, 5, 7 days, respectively. We systematically characterized 3,609 proteins, 1,308 acetylation sites, and 1,947 succinylation sites across multiple storage time points (D1, D3, D5, D7). We distinct temporal patterns of post-translational modifications, with succinylation showing more extensive coverage than acetylation in platelets. Pathway enrichment analysis revealed extensive metabolic reprogramming involving complement activation, energy metabolism, and cellular detoxification processes. The identification of specific motif patterns provided mechanistic insights into the functional specificity of these modifications. Random forest machine learning identified 20 core regulatory proteins representing critical nodes in PSL development. Furthermore, we employed real - time quantitative polymerase chain reaction (RT - QPCR) to measure the expression levels of key genes related to platelet function and PTM - associated pathways. By mapping the interplay between proteomic abundance shifts and PTM dynamics, this study provides a multidimensional understanding of PSL, establishing a foundational framework for optimizing storage protocols and enhancing transfusion safety.

Purpose:To provide the first insight into the proteomic dynamics during platelet storage.Experiment design: In this study, based on TMT-labeled LC-MS/MS analysis, combined with antibody-affinity enrichment and purification for acetylated and succinylated peptides, we performed quantification of global proteomics, acetylome and succinylome.Simultaneously, dynamic molecular changes and functional transformation of platelet were also characterized under proper conditions stored for 1, 3, 5, 7 days, respectively.Results:3,100 proteins are quantified from a total of 3,609 proteins identified from platelets. Out of 1,308 acetylated sites identified in 648 proteins, 790 sites in 396 proteinsare quantifiable. There are 1,947 succinylated sites in 959 proteins in which 1,279 sites in 661 proteins are quantifiable.We screened the differential expression changes of global proteins, acetyl- and succinyl- proteins, and systematically interpreted their molecular functions, biological processes, cellular components, pathways and motif characters to fully investigate the molecular dynamics and biological functions of platelets. Conclusions and clinical relevance:This paper is the first systematic exploration of proteomes and modified proteomes of platelet dynamics during storage in the aim to improve our understanding of platelet biology, which may be a valuable reference for further research and clinical application.

Cancer-associated fibroblasts (CAFs) are a major stromal component of the tumor microenvironment (TME) and actively participate in tumor progression. Altho