Department of Electrical Engineering, University of Washington , Seattle, Washington 98195, United States.
Department of Computer Science & Engineering, University of Washington , Seattle, Washington 98195, United States.
J Am Chem Soc. 2016 Apr 20;138(15):5076-86. doi: 10.1021/jacs.6b00277. Epub 2016 Apr 11.
Even a single-nucleotide difference between the sequences of two otherwise identical biological nucleic acids can have dramatic functional consequences. Here, we use model-guided reaction pathway engineering to quantitatively improve the performance of selective hybridization probes in recognizing single nucleotide variants (SNVs). Specifically, we build a detection system that combines discrimination by competition with DNA strand displacement-based catalytic amplification. We show, both mathematically and experimentally, that the single nucleotide selectivity of such a system in binding to single-stranded DNA and RNA is quadratically better than discrimination due to competitive hybridization alone. As an additional benefit the integrated circuit inherits the property of amplification and provides at least 10-fold better sensitivity than standard hybridization probes. Moreover, we demonstrate how the detection mechanism can be tuned such that the detection reaction is agnostic to the position of the SNV within the target sequence. in contrast, prior strand displacement-based probes designed for kinetic discrimination are highly sensitive to position effects. We apply our system to reliably discriminate between different members of the let-7 microRNA family that differ in only a single base position. Our results demonstrate the power of systematic reaction network design to quantitatively improve biotechnology.
即使两种在序列上完全相同的生物核酸之间只有一个核苷酸的差异，也可能会产生巨大的功能后果。在这里，我们使用模型引导的反应途径工程来定量提高选择性杂交探针识别单核苷酸变体 (SNV) 的性能。具体来说，我们构建了一个检测系统，该系统结合了竞争的区分与基于 DNA 链置换的催化扩增。我们从理论和实验两方面证明，这种系统在与单链 DNA 和 RNA 结合时的单核苷酸选择性比单纯的竞争杂交的区分要好两倍。作为额外的好处，集成电路继承了放大的特性，比标准杂交探针的灵敏度至少提高了 10 倍。此外，我们还展示了如何调整检测机制，使检测反应对目标序列中 SNV 的位置不敏感。相比之下，先前设计用于动力学区分的基于链置换的探针对位置效应非常敏感。我们将我们的系统应用于可靠地区分仅在一个碱基位置上不同的 let-7 microRNA 家族的不同成员。我们的结果证明了系统反应网络设计在定量提高生物技术方面的强大功能。
