Selective rolling circle amplification detection of site-specific DNA 5-hydroxymethylcytosine

Site-specific 5-hydroxymethylcytosine (5hmC) has been identified as a potential biomarker involving diagnosis, prognosis, and tumor immune microenvironment across multiple cancers. However, directly distinguishing trace levels of 5hmC from its highly abundant cytosine/5-methylcytosine (C/5mC) analogs remains a technical challenge. Herein, we present a 5hmC selective ligation-rolling circle amplification (5hmC-RCA) that enables the direct, high-specificity discrimination of 5hmC from C/5mC. The high specificity of 5hmC-RCA depends on the high selectivity of HiFi Taq DNA Ligase, which exhibits robust tolerance for mismatched 5hmC/A base pairs, thereby efficiently ligating the associated nicking site. While the mismatched 5mC/A or C/A pairs fail to be recognized by HiFi Taq DNA Ligase. As a proof of principle, the 5hmC-RCA assay was evaluated for its performance using Septin9 gene, which considered as key biomarkers in the prognosis and diagnosis of CRC, as the target, demonstrating a detection sensitivity of approximately 0.5%. Notably, the assay exhibited no cross-reactivity with unmodified target even at a high background concentration of 5,000 copies per reaction. By coupling HiFi Taq DNA Ligase-mediated high selective ligation with RCA, sensitive and selective quantification of site-specific 5hmC is achieved. Due to the simplicity of 5hmC-RCA, it also has potential for use in cfDNA originating from the tumor microenvironment and immune cells.