Tao Shurui, Long Yi, Liu Guozhen
CUHKSZ-Boyalife Regenerative Medicine Engineering Joint Laboratory, School of Medicine, The Chinese University of Hong Kong, Shenzhen 518172, China.
Integrated Devices and Intelligent Diagnosis (ID2) Laboratory, Ciechanover Institute of Precision and Regenerative Medicine, School of Medicine, The Chinese University of Hong Kong, Shenzhen 518172, China.
Biosensors (Basel). 2024 Dec 15;14(12):618. doi: 10.3390/bios14120618.
The Phi29 DNA polymerase is renowned for its processivity in synthesizing single-stranded DNA amplicons by rolling around a circularized DNA template. However, DNA synthesis rolling circle amplification (RCA) is significantly hindered by the secondary structure in the circular template. To overcome this limitation, an engineered circular template without secondary structure could be utilized to improve the sensitivity of RCA-based assays without increasing its complexity. We herein proposed an entropy-driven special RCA technology for the detection of HPV16 E7 gene at room temperature. The strategy is composed of a molecular beacon containing a loop region for nucleic acid target recognition and a stem region to initiate RCA. With the target analyte, the stem region of the molecular beacon will be exposed and then hybridized with a special circular template to initiate the DNA amplification. We tested different designs of the molecular beacon sequence and optimized the assay's working conditions. The assay achieved a sensitivity of 1 pM in 40 min at room temperature. The sensitivity of this assay, at 1 pm, is about a hundred-fold greater than that of conventional linear RCA performed in solution. Our proposed sensor can be easily reprogrammed for detecting various nucleic acid markers by altering the molecular beacon's loop. Its simplicity, rapid assay time, and low cost make it superior to RCA sensors that utilize similar strategies.
Phi29 DNA聚合酶以其通过围绕环形化DNA模板滚动来合成单链DNA扩增子的持续合成能力而闻名。然而，DNA合成滚环扩增（RCA）受到环形模板二级结构的显著阻碍。为了克服这一限制，可以利用一种没有二级结构的工程化环形模板来提高基于RCA的检测灵敏度，而不增加其复杂性。我们在此提出一种熵驱动的特殊RCA技术，用于在室温下检测HPV16 E7基因。该策略由一个分子信标组成，该分子信标包含一个用于核酸靶标识别的环区域和一个用于启动RCA的茎区域。在存在靶分析物的情况下，分子信标的茎区域将被暴露，然后与一个特殊的环形模板杂交以启动DNA扩增。我们测试了分子信标序列的不同设计，并优化了检测的工作条件。该检测在室温下40分钟内实现了1 pM的灵敏度。该检测在1 pM时的灵敏度比在溶液中进行的传统线性RCA高出约一百倍。我们提出的传感器可以通过改变分子信标的环轻松重新编程以检测各种核酸标记物。其简单性、快速的检测时间和低成本使其优于采用类似策略的RCA传感器。
