Institute of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489, Berlin, Germany.
Chemistry. 2021 Oct 13;27(57):14189-14194. doi: 10.1002/chem.202102600. Epub 2021 Sep 13.
Fluorogenic hybridization probes allow the detection of RNA and DNA sequences in homogeneous solution. Typically, one target molecule activates the fluorescence of a single probe molecule. This limits the sensitivity of nucleic acid detection. Herein, we report a self-immolative molecular beacon (iMB) that escapes the one-target/one-probe paradigm. The iMB probe includes a photoreductively cleavable N-alkyl-picolinium (NAP) linkage within the loop region. A fluorophore at the 5'-end serves, on the one hand, as a reporter group and, on the other hand, as a photosensitizer of a NAP-linker cleavage reaction. In the absence of target, the iMB adopts a hairpin shape. Quencher groups prevent photo-induced cleavage. The iMB opens upon hybridization with a target, and both fluorescent emission as well as photo-reductive cleavage of the NAP linker can occur. In contrast to previous chemical amplification reactions, iMBs are unimolecular probes that undergo cleavage leading to products that have lower target affinity than the probes before reaction. Aided by catalysis, the method allowed the detection of 5 pm RNA target within 100 min.
荧光杂交探针可在均相溶液中检测 RNA 和 DNA 序列。通常，一个靶标分子仅能激活单个探针分子的荧光。这限制了核酸检测的灵敏度。在此，我们报告了一种自耗散分子信标 (iMB)，它摆脱了单靶标/单探针的模式。iMB 探针在环区包含一个光还原裂解的 N-烷基吡啶鎓 (NAP) 键。5'-端的荧光团一方面作为报告基团，另一方面作为 NAP-连接体裂解反应的光敏剂。在没有靶标的情况下，iMB 采用发夹形状。猝灭基团防止光诱导裂解。iMB 与靶标杂交后打开，荧光发射和 NAP 连接体的光还原裂解均可发生。与以前的化学扩增反应不同，iMB 是单分子探针，其裂解导致产物与反应前的探针相比对靶标具有更低的亲和力。在催化作用的辅助下，该方法能够在 100 分钟内检测到 5 pm 的 RNA 靶标。
