Department of Chemical and Biological Engineering, ‡Renewable and Sustainable Energy Institute (RASEI), §BioFrontiers Institute, and ⊥Materials Science and Engineering, University of Colorado Boulder , Boulder, Colorado 80309, United States.
ACS Nano. 2017 Nov 28;11(11):11169-11181. doi: 10.1021/acsnano.7b05500. Epub 2017 Oct 6.
Several nanoscale electronic methods have been proposed for high-throughput single-molecule nucleic acid sequence identification. While many studies display a large ensemble of measurements as "electronic fingerprints" with some promise for distinguishing the DNA and RNA nucleobases (adenine, guanine, cytosine, thymine, and uracil), important metrics such as accuracy and confidence of base calling fall well below the current genomic methods. Issues such as unreliable metal-molecule junction formation, variation of nucleotide conformations, insufficient differences between the molecular orbitals responsible for single-nucleotide conduction, and lack of rigorous base calling algorithms lead to overlapping nanoelectronic measurements and poor nucleotide discrimination, especially at low coverage on single molecules. Here, we demonstrate a technique for reproducible conductance measurements on conformation-constrained single nucleotides and an advanced algorithmic approach for distinguishing the nucleobases. Our quantum point contact single-nucleotide conductance sequencing (QPICS) method uses combed and electrostatically bound single DNA and RNA nucleotides on a self-assembled monolayer of cysteamine molecules. We demonstrate that by varying the applied bias and pH conditions, molecular conductance can be switched ON and OFF, leading to reversible nucleotide perturbation for electronic recognition (NPER). We utilize NPER as a method to achieve >99.7% accuracy for DNA and RNA base calling at low molecular coverage (∼12×) using unbiased single measurements on DNA/RNA nucleotides, which represents a significant advance compared to existing sequencing methods. These results demonstrate the potential for utilizing simple surface modifications and existing biochemical moieties in individual nucleobases for a reliable, direct, single-molecule, nanoelectronic DNA and RNA nucleotide identification method for sequencing.
已经提出了几种用于高通量单分子核酸序列识别的纳米级电子方法。虽然许多研究都显示了大量的测量结果作为“电子指纹”，并且在区分 DNA 和 RNA 碱基（腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶和尿嘧啶）方面具有一定的潜力，但碱基调用的准确性和置信度等重要指标远低于当前的基因组方法。一些问题，如不可靠的金属-分子结形成、核苷酸构象的变化、负责单核苷酸传导的分子轨道之间的差异不足，以及缺乏严格的碱基调用算法，导致重叠的纳米电子测量和核苷酸识别能力较差，尤其是在单分子的低覆盖率下。在这里，我们展示了一种在构象受限的单核苷酸上进行可重复电导测量的技术，以及一种用于区分碱基的高级算法方法。我们的量子点接触单核苷酸电导测序（QPICS）方法使用梳状和静电束缚的单个 DNA 和 RNA 核苷酸，在半胱氨酸分子的自组装单层上。我们证明，通过改变施加的偏压和 pH 条件，可以打开和关闭分子电导，从而导致电子识别的核苷酸可逆扰动（NPER）。我们利用 NPER 作为一种方法，在低分子覆盖率（约 12×）下，使用 DNA/RNA 核苷酸的无偏单测量，实现了 DNA 和 RNA 碱基调用的>99.7%的准确性，与现有测序方法相比，这是一个重大进展。这些结果表明，利用单个碱基中的简单表面修饰和现有生化基团，为可靠、直接、单分子、纳米电子 DNA 和 RNA 核苷酸识别方法用于测序，具有很大的潜力。
