High‐Fidelity Profiling of Multiple Nearby Mutations via Cooperative Recognition

Abstract Detection methods with single‐nucleotide specificity are essential tools for nucleic acid analysis in diverse clinical and biological settings. However, both hybridization‐based and enzyme‐based methods are only effective for discriminating single‐nucleotide mutations at certain positions, making it difficult to detect nucleic acid targets having multiple nearby mutations. Herein, we describe the design of cooperative recognition probes (CRPs) that integrate both hybridization and ligation‐based recognition mechanisms and thus are highly effective for discriminating mutations throughout all positions. The cooperative nature of CRPs further enables AND‐gate‐based detection of multiple nearby mutations with high fidelity and specificity. Moreover, CRPs generate circular or linear ligation products that can be readily amplified by rolling circle amplification or polymerase chain reaction, making our strategy readily adaptable to diverse biological and clinical settings. Leveraging CRPs, we demonstrate the detection of nucleic acid targets that are difficult to be discriminated using conventional strategies, such as the highly specific discrimination of microRNA from its family members and isoforms, and the high‐fidelity identification of drug‐resistant single‐nucleotide variants in the presence of nearby synchronous mutations in lung cancer samples.