Construction of a multiple ligation-driven exponentially symmetric T7-transcription machinery for single-molecule monitoring of diverse single-nucleotide polymorphisms in human cancers

邻近连接试验 单核苷酸多态性 核苷酸 结扎 抄写(语言学) 生物 分子生物学 转录因子 发起人 遗传学 化学 计算生物学 基因 基因型 基因表达 哲学 受体 语言学
作者
Lijuan Wang,Xiao-wen Liu,Xinyi Zhang,Yun Han,Qian Liu,Ziyue Wang,Chun‐yang Zhang
出处
期刊:Chemical Engineering Journal [Elsevier BV]
卷期号:480: 148251-148251 被引量:5
标识
DOI:10.1016/j.cej.2023.148251
摘要

Single nucleotide polymorphism (SNP) is one of the most abundant genetic variations among individuals. Since one human disease is generally associated with several SNPs, simultaneous monitoring of diverse SNPs is particularly important. Herein, we demonstrate the construction of a multiple ligation-driven exponentially symmetric T7-transcription machinery for single-molecule monitoring of diverse SNPs. In presence of KRAS-135C and BRAF-V600E, ligation probes (i.e., LP-1 and LP-2) and template probes (i.e., TP-1-TP-2 duplex) are splinted together to construct transcription templates 1 and 2, and their corresponding T7 promoters will activate symmetric transcription amplifications to produce two reporter probes (i.e., RP-1 and RP-2). RP-1 and RP-2 selectively hybridize with signal probes (i.e., SP-1 and SP-2) to initiate cyclic degradation of SP-1 and SP-2 for liberating Cy3 and Cy5 as well as RP-1 and RP-2. Subsequently, RP-1 and RP-2 act as the ligation templates to splint LP-1/LP-2 and TP-1-TP-2 together, initiating new cycles of ligation-transcription-degradation for exponential accumulation of Cy3 and Cy5. KRAS-135C and BRAF-V600E mutants can be quantitatively detected by simply counting Cy3 and Cy5 molecules. This nanomachine exhibits a limit of detection (LOD) of 7.24 × 10−20 M for KRAS-135C and 3.72 × 10−20 M for BRAF-V600E with a dynamic range of 9 orders of magnitude. Moreover, it can differentiate SNPs with sequence homology, distinguish rare SNPs with 0.01 % mutation level, discriminate SNPs in cancer cells and normal cells, and even quantify SNPs in various cancer cells at single-cell level, providing a universal platform for genomic SNP-related biomedical studies and molecular diagnostics.
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