纳米孔道单分子检测结直肠癌MicroRNAs
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摘要
MicroRNA(miRNA)可用于癌症的早期诊断、预后判断,其分析检测具有重要临床意义.结直肠癌的发生、发展与miRNA 21、miRNA 92等的异常表达明显相关.本研究设计了以poly(d T)n为引导链的DNA探针(probe)并尝试使用α-溶血素(α-HL)纳米孔道单分子检测方法检测结直肠癌miRNAs.miRNA·probe复合物分子穿过α-HL纳米孔道限域空间时,由于probe链长、序列不同导致probe-α-HL相互作用不同,miRNA 92·probe 92、miRNA 21·probe 21、miRNA 16·probe 16输出为形态、阻断时间不同的多台阶特征信号,实现了三种miRNAs的有效区分.实验证明,此方法可以用于检测血清实际样品.因此,未来有望使用α-HL构建miRNA超灵敏单分子生物传感器.
MicroRNAs(miRNAs), 18~22 nucleotides in length, are a class of single-strand noncoding short RNAs and have been used as biomarkers for diagnosis and prognosis of cancers. Herein, an α-hemolysin(α-HL) nanopore was adapted for the colorectal cancer-associated miRNAs analysis, with the merits of high-throughput, ultra-sensitivity and no requirements of amplification/labelling. DNA probes, consisting of a signal tag in each end and a response element in the middle section, were designed. The response element could be well-matched with miRNA and utilized for specific recognition of the target miRNA, while the signal tag increased the capture rate of the miRNA·probe complex. Due to the poor stacking of thymine residues, poly(d T)n need to overcome a high entropic barrier when traversing through the α-HL nanopore confined space, resulting in distinct double-level blocked events, which contributes to the visualized differences in signal shape and prolonged duration. Thus, poly(d T)n was selected as the signal tag of probe. Added in the cis side of α-HL, miRNA·probe was forced to traverse across the nanopore confined space under the potential of 140 m V through a pair of Ag/Ag Cl electrodes(cis grounded). Typical three-stage blocked event was observed, reflecting the translocation process: capture and dissociation of miRNA·probe, translocation of probe, temporarily residence and translocation of miRNA. Stage 1(S1) represented the process from capture of miRNA·probe complex to translocation of the entire probe. The typical blocked events of miRNA 92·probe 92 showed a two-level S1, where Level 1(L1) with a current blockage of 0.57±0.01 was generated mainly by translocation of the poly(d T)40 signal tag. As the duration is associated with DNA length, probe 21 with smaller poly(d T)20 signal tag was designed to detect miRNA 21, resulting in a shorter L1 of miRNA 21·probe 21 whose duration(t D-L1) was 1/3 of that for miRNA 92·probe 92. As the signal shapes vary with DNA sequences, probe 16 with signal tag of poly(d C)40 was used to sense miRNA 16, with miRNA 16·probe 16 producing a different single-level S1 with miRNA 92·probe 92 and miRNA 21·probe 21. The statistical results demonstrated that the three kinds of miRNA·probe produced different durations for S1(t D-S1), possibly indicating the differences in probe-α-HL interaction. Therefore, miRNA 92, miRNA 21 and miRNA 16 could be well identified by t D-L1(signal shape) and t D-S1(duration). Moreover, the serum sample have been tested. Hence, α-HL nanopore can be applied to build ultrasensitive single molecule biosensor for miRNA.
MicroRNAs(miRNAs), 18~22 nucleotides in length, are a class of single-strand noncoding short RNAs and have been used as biomarkers for diagnosis and prognosis of cancers. Herein, an α-hemolysin(α-HL) nanopore was adapted for the colorectal cancer-associated miRNAs analysis, with the merits of high-throughput, ultra-sensitivity and no requirements of amplification/labelling. DNA probes, consisting of a signal tag in each end and a response element in the middle section, were designed. The response element could be well-matched with miRNA and utilized for specific recognition of the target miRNA, while the signal tag increased the capture rate of the miRNA·probe complex. Due to the poor stacking of thymine residues, poly(d T)n need to overcome a high entropic barrier when traversing through the α-HL nanopore confined space, resulting in distinct double-level blocked events, which contributes to the visualized differences in signal shape and prolonged duration. Thus, poly(d T)n was selected as the signal tag of probe. Added in the cis side of α-HL, miRNA·probe was forced to traverse across the nanopore confined space under the potential of 140 m V through a pair of Ag/Ag Cl electrodes(cis grounded). Typical three-stage blocked event was observed, reflecting the translocation process: capture and dissociation of miRNA·probe, translocation of probe, temporarily residence and translocation of miRNA. Stage 1(S1) represented the process from capture of miRNA·probe complex to translocation of the entire probe. The typical blocked events of miRNA 92·probe 92 showed a two-level S1, where Level 1(L1) with a current blockage of 0.57±0.01 was generated mainly by translocation of the poly(d T)40 signal tag. As the duration is associated with DNA length, probe 21 with smaller poly(d T)20 signal tag was designed to detect miRNA 21, resulting in a shorter L1 of miRNA 21·probe 21 whose duration(t D-L1) was 1/3 of that for miRNA 92·probe 92. As the signal shapes vary with DNA sequences, probe 16 with signal tag of poly(d C)40 was used to sense miRNA 16, with miRNA 16·probe 16 producing a different single-level S1 with miRNA 92·probe 92 and miRNA 21·probe 21. The statistical results demonstrated that the three kinds of miRNA·probe produced different durations for S1(t D-S1), possibly indicating the differences in probe-α-HL interaction. Therefore, miRNA 92, miRNA 21 and miRNA 16 could be well identified by t D-L1(signal shape) and t D-S1(duration). Moreover, the serum sample have been tested. Hence, α-HL nanopore can be applied to build ultrasensitive single molecule biosensor for miRNA.
引文
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[19]Wang,Y.;Zheng,D.-L.;Tan,Q.-L.;Wang,M.X.;Gu,L.-Q.Nat.Nanotechnol.2011,6,668.
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[24]Gu,Z.;Ying,Y.-L.;Cao,C.;He,P.-G.;Long,Y.-T.Anal.Chem.2015,87,907.