School of Printing and Packaging, Wuhan University, Wuhan, 430079, P. R. China.
The Centre of Analysis and Measurement of Wuhan University, Wuhan University, Wuhan, 430072, P. R. China.
Small. 2021 Jan;17(2):e2006370. doi: 10.1002/smll.202006370. Epub 2020 Dec 16.
The analysis of mutant nucleic acid (NA) variants can provide crucial clinical and biological insights for many diseases. Yet, existing analysis techniques are generally constrained by nonspecific "noise" signals from excessive wildtype background sequences, especially under rapid isothermal multiplexed target amplification conditions. Herein, the molecular hybridization chemistry between NA bases is manipulated to suppress noise signals and achieve ultraselective multiplexed detection of cancer gene fusion NA variants. Firstly, modified locked NA (LNA) bases are rationally introduced into oligonucleotide sequences as designed "locker probes" for high affinity hybridization to wildtype sequences, leading to enrichment of mutant variants for multiplexed isothermal amplification. Secondly, locker probes are coupled with a customized "proximity-programmed" (SERS) readout which allows precise control of hybridization-based plasmonic signaling to specifically detect multiple target amplicons within a single reaction. Moreover, the use of triple bond Raman reporters endows NA noise signal-free quantification in the Raman silent region (≈1800-2600 cm ). With this dual molecular hybridization-based strategy, ultraselective multiplexed detection of gene fusion NA variants in cancer cellular models is actualized with successful noise suppression of native wildtype sequences. The distinct benefits of isothermal NA amplification and SERS multiplexing ability are simultaneously harnessed.
突变核酸 (NA) 变体的分析可为许多疾病提供关键的临床和生物学见解。然而，现有的分析技术通常受到来自过多野生型背景序列的非特异性“噪声”信号的限制，尤其是在快速等温多重靶扩增条件下。在此，通过操纵 NA 碱基之间的分子杂交化学来抑制噪声信号，实现癌症基因融合 NA 变体的超选择性多重检测。首先，将经过合理修饰的锁核酸 (LNA) 碱基作为设计的“锁探针”引入寡核苷酸序列中，用于与野生型序列进行高亲和力杂交，从而富集用于多重等温扩增的突变变体。其次，锁探针与定制的“临近程序化”（SERS）读出相偶联，允许对基于杂交的等离子体信号进行精确控制，以在单个反应中特异性检测多个靶扩增子。此外，三键拉曼报告基团的使用赋予了在拉曼静默区（≈1800-2600cm）进行无 NA 噪声信号定量的能力。通过这种基于双重分子杂交的策略，在癌症细胞模型中实现了基因融合 NA 变体的超选择性多重检测，并成功抑制了天然野生型序列的噪声。同时利用了等温 NA 扩增和 SERS 多重化能力的优势。
