Rolling circle amplification for next-generation molecular diagnostics, genome analysis, and spatial transcriptome...

Rolling circle amplification (RCA) has emerged as a highly versatile and robust isothermal amplification technology, offering exceptional sensitivity, specificity, and scalability for next-generation molecular diagnostics and multi-omics research. Its ability to generate long, repetitive DNA sequences with high fidelity has made it a pivotal tool in disease diagnostics, genomic analysis, and spatial transcriptome profiling. Recent advancements have expanded RCA into various formats, including solution-phase, solid-phase, hydrogel-based, and digital RCA, enhancing its analytical performance and adaptability across diverse biological applications. The integration of RCA with CRISPR-based detection, biosensors, and nanoparticle-assisted signal amplification has significantly improved its diagnostic potential, enabling ultrasensitive detection of nucleic acids, proteins, extracellular vesicles, and single cells. In addition to diagnostics, RCA has profoundly impacted genomic research, particularly in extrachromosomal circular DNA sequencing, offering insights into genome instability and cancer evolution. In spatial transcriptomics, RCA-based in situ RNA sequencing and hybridization have enabled high-resolution gene expression mapping in tissue architecture, providing invaluable insights into cellular heterogeneity and disease microenvironments. This review comprehensively explores the fundamental principles, design modifications, and advanced signal amplification strategies of RCA, with a focus on its applications in molecular diagnostics, genome analysis, and spatial transcriptome profiling. Additionally, it addresses key challenges, including reaction specificity, automation, and cost-effectiveness, and discusses future directions for integrating RCA into high-throughput molecular diagnostics, biomarker discovery, and single-cell and spatial multi-omics platforms. Through continuous innovation, RCA is poised to play a transformative role in precision medicine, next-generation sequencing, and biomedical research.