Chemical tags facilitate the sensing of individual DNA strands with nanopores
Chemical tags facilitate the sensing of individual DNA strands with nanopores
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DOI：
10.1002/anie.200800183
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Howorka, Stefan
中科院分区：
文献类型：
--
作者：
Mitchell, Nick;Howorka, Stefan
纳米孔记录是一种电分析技术，其中单个分子阻塞纳米级孔并引起离子电流的可检测调制。[1-5]单分子方法已被用于分析蛋白质，毒素，金属离子，药物分子和双链DNA中的单点突变。[6-11]尽管在检测分离的核苷酸[12]和静态DNA链中的单碱基位置方面取得了进展[13]，但到目前为止，还不可能检测单个链中的多个碱基。实现这一目标的主要技术障碍之一是电压驱动的DNA链通过孔的高速;这导致分析分辨率不足。在此，我们提出了一种新的方法，可以减慢单链DNA（ssDNA）的速度，并能够检测多个单独的碱基。我们表明，化学标签连接到基地造成空间阻滞每次修改基地通过一个狭窄的孔。所得到的特征电流签名对于标签的化学成分和尺寸是特定的。我们的方法与修饰的DNA检测是独立的孔工程，并有可能被应用到广泛的固态纳米孔，以扩展其传感库。[2，3，14]这是一种新的策略，因为通过分析物分子的化学修饰而不是孔的工程化来促进检测。使用蛋白孔α-溶血素的非工程化版本（αHL;图1A）测试了这种用于DNA碱基特异性鉴定的新方法。αHL孔在过去已被广泛用于感测未修饰的RNA和DNA链。[1，15]因此，它构成了用化学修饰的DNA进行感测的良好参考点。αHL具有明确的结构，狭窄内缩处的管腔直径为1.3 nm，孔反侧β桶中的管腔直径为2 nm（图1A）。[16]已知平均横截面直径为0.9-1.2 nm的ssDNA [17]可以通过内缢痕。[1，18]我们推测，附着在单独碱基上的化学标签会增加DNA的横截面直径，因此每次修饰的碱基通过窄孔收缩时都会减慢易位。为了测试该方法，使用由市售DNA寡核苷酸和由肽组成的标签组成的模型系统。选择肽是因为它们的大小，长度，电荷和疏水性可以很容易地以模块化的方式进行调整，以优化孔blockades.We首次证实，一个单一的肽标签是能够延缓链易位。DNA链寡核苷酸O 1，长度为27个碱基，在内部碱基处用六聚组氨酸标签H6 C1修饰（图1B;见支持性信息）。在纳米孔记录中分析所得肽-DNA缀合物H6 C1-O 1。在不存在DNA的情况下，当在反式侧施加正电位时，野生型αHL孔产生的电导为（1900+ 120）pS（独立记录数n= 4）（图1C和A）。将未修饰的寡核苷酸O 1添加到孔的顺式侧（图1A）导致短的高振幅事件（图1D）。其特征在于，相对于正常人，平均振幅为（91.7 ± 1.1）%。
Nanopore recording is an electrical analytical technique in which individual molecules block a nanometer-scale pore and cause detectable modulations in ionic current.[1–5] The singlemolecule approach has been exploited to analyze proteins, toxins, metal ions, drug molecules, and single-point mutations in double-stranded DNA.[6–11] Despite progress in the sensing of isolated nucleotides [12] and single base positions in static DNA strands,[13] it has, so far, not been possible to detect multiple bases in an individual strand. One of the main technical hurdles towards this aim is the high speed at which the voltage-driven DNA strands pass through the pore; this leads to insufficient analytical resolution. Herein, we present a new approach that slows down single-stranded DNA (ssDNA) and enables the detection of multiple separate bases. We show that chemical tags attached to bases cause a steric blockade each time a modified base passes through a narrow pore. The resulting characteristic current signatures are specific for the chemical composition and the size of the tags. Our approach for detection with modified DNA is independent of pore engineering and can potentially be applied to a wide range of solid-state nanopores to extend their sensing repertoire.[2, 3, 14] This is a novel strategy because the detection is facilitated by the chemical modification of the analyte molecules rather than the engineering of pores. This new approach for the base-specific identification of DNA was tested with the nonengineered version of the protein pore α-hemolysin (αHL; Figure 1A). The αHL pore has been widely used in the past for the sensing of unmodified RNA and DNA strands.[1, 15] It therefore constitutes a good reference point for sensing with chemically modified DNA. αHL is of defined architecture with a lumen diameter of 1.3 nm at the narrow inner constriction and of 2 nm in the β barrel at the trans side of the pore (Figure 1A).[16] ssDNA with an average cross-sectional diameter of 0.9–1.2 nm [17] is known to pass the inner constriction.[1, 18] We speculated that chemical tags attached to separate bases would increase the cross-sectional diameter of the DNA and hence slow down translocation each time a modified base passes the narrow pore constriction. To test the approach, a model system consisting of commercially available DNA oligonucleotides and tags composed of peptides was used. Peptides were selected because their size, length, charge, and hydrophobicity can be easily tuned in a modular fashion to optimize pore blockades.We first confirmed that a single peptide tag is capable of retarding strand translocation. DNA-strand oligonucleotide O1, of 27 bases in length, was modified with the hexahistidine tag H6C1 at an internal base (Figure 1B; see the Supporting Information). The resulting peptide–DNA conjugate, H6C1–O1, was analyzed in nanopore recordings. In the absence of DNA, the wild-type αHL pore yielded a conductance of (1900+ 120) pS (number of independent recordings n= 4) when a positive potential was applied at the trans side (Figure 1C and A). The addition of unmodified oligonucleotide O1 to the cis side of the pore (Figure 1A) led to short high-amplitude events (Figure 1 D). These were characterized by an average amplitude of (91.7+ 1.1)% relative to the