MiRNAs are known to be involved in a series of diseases, including breast cancer, and they have the potential to serve as diagnostic/prognostic markers and therapeutic targets. A prerequisite for miRNAs to be applied in clinical practice is the quantitative profiling of their expression. However, the majority of current assays used in miRNA detection are highly enzyme-dependent. In this study, a novel enzyme-free assay was developed that relies on stacking hybridization and a photocleavable DNA-PL-peptide probe, which contains a reporter peptide (AVLGVDPFR), a photocleavable o-nitrobenzyl derivative linker and a detection DNA sequence that is complementary to a part of the target miRNA (e.g., miR-21, miR-125a or miR-200c). Stacking hybridization enabled the DNA-PL-peptide probe to capture DNA in a contiguous tandem arrangement to generate a long DNA single strand complementary to the target miRNA. Then, photolysis was initiated to rapidly release the reporter peptide, and the reporter peptide was ultimately monitored by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this experiment, the parameters linked with photorelease, binding, conjugation and hybridization were characterized. The results showed that the assay time was significantly shortened, and the detection specificity was improved. After validation of the assay, the target miRNA level was determined in human breast cells and tissue samples. The results demonstrated that photocleavable materials coupled with mass spectrometric detection have great potential in clinical practice.

中文翻译：

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可光裂解的质谱DNA肽探针可实现对microRNA的快速，特异性无酶检测。

已知MiRNA与包括乳腺癌在内的一系列疾病有关，它们有潜力用作诊断/预后标志物和治疗靶标。miRNA在临床实践中应用的先决条件是其表达的定量分析。但是，目前在miRNA检测中使用的大多数检测方法高度依赖酶。在这项研究中，开发了一种新颖的无酶测定方法，该方法依赖于堆叠杂交和可光裂解的DNA-PL-肽探针，该探针包含一个报告肽（AVLGVDPFR），一个可光裂解的邻硝基苄基衍生物接头和一个可检测的DNA序列。与部分靶标miRNA（例如，miR-21，miR-125a或miR-200c）互补。堆叠杂交使DNA-PL-肽探针能够以连续串联的方式捕获DNA，从而生成与目标miRNA互补的长DNA单链。然后，开始光解以快速释放报告肽，并最终通过液相色谱-串联质谱（LC-MS / MS）监测报告肽。在该实验中，表征了与光释放，结合，结合和杂交相关的参数。结果表明，测定时间明显缩短，检测特异性提高。验证分析后，确定人乳腺细胞和组织样品中的目标miRNA水平。结果表明，光可裂解材料结合质谱检测在临床实践中具有巨大潜力。