A guide to accelerated direct digital counting of single nucleic acid molecules by FRET-based intramolecular kinetic fingerprinting.
A guide to accelerated direct digital counting of single nucleic acid molecules by FRET-based intramolecular kinetic fingerprinting.
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DOI：
10.1016/j.ymeth.2021.06.014
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发表时间：
2022-01
期刊：
影响因子：
--
通讯作者：
Walter NG
中科院分区：
文献类型：
--
作者：
Mandal S;Khanna K;Johnson-Buck A;Walter NG
存在于体液中的无细胞核酸（cfNA）如短的非编码microRNA（miRNA）和循环肿瘤DNA（ctDNA）已成为潜在的癌症生物标志物。因此，用于快速、高度特异性和灵敏地监测生物流体中cfNA的方法作为临床诊断工具变得越来越有吸引力。作为下一代技术，我们提供了一个实用的指南，用于无扩增，单分子Förster共振能量转移（smFRET）为基础的动力学指纹方法，称为通过平衡泊松采样的分子内单分子识别，或iSiMREPS，用于快速检测和计数miRNA和突变ctDNA，具有几乎无限的特异性和单分子灵敏度。iSiMREPS利用一对荧光检测探针，其中一个探针将靶分子固定在表面上，另一个探针瞬时可逆地结合靶以产生特征性时间分辨指纹作为在全内反射荧光显微镜中检测的smFRET信号。对这些动力学指纹的分析使得能够在与靶分子的特异性结合和非特异性背景结合之间进行近乎完美的区分。通过使用变性剂甲酰胺加速动力学指纹分析，并通过支点介导的链置换从表面去除无靶标探针来减少背景信号，iSiMREPS已被证明可计数miR-141和EGFR外显子19缺失ctDNA分子，检测限（LOD）分别为约1和3 fM，以及低至0.0001%的突变等位基因分数，在每个视场仅约10 s的标准采集时间内。在这篇综述中，我们为在研究和临床诊断中更广泛地实施iSiMREPS提供了详细的路线图，结合了快速分析，高特异性和高灵敏度。
Cell-free nucleic acids (cfNAs) such as short non-coding microRNA (miRNA) and circulating tumor DNA (ctDNA) that reside in bodily fluids have emerged as potential cancer biomarkers. Methods for the rapid, highly specific, and sensitive monitoring of cfNAs in biofluids have, therefore, become increasingly attractive as clinical diagnosis tools. As a next generation technology, we provide a practical guide for an amplification-free, single molecule Förster resonance energy transfer (smFRET)-based kinetic fingerprinting approach termed intramolecular single molecule recognition through equilibrium Poisson sampling, or iSiMREPS, for the rapid detection and counting of miRNA and mutant ctDNA with virtually unlimited specificity and single molecule sensitivity. iSiMREPS utilizes a pair of fluorescent detection probes, wherein one probe immobilizes the target molecules on the surface, and the other probe transiently and reversibly binds to the target to generate characteristic time-resolved fingerprints as smFRET signal that are detected in a total internal reflection fluorescence microscope. Analysis of these kinetic fingerprints enables near-perfect discrimination between specific binding to target molecules and nonspecific background binding. By accelerating kinetic fingerprinting using the denaturant formamide and reducing background signals by removing target-less probes from the surface via toehold-mediated strand displacement, iSiMREPS has been demonstrated to count miR-141 and EGFR exon 19 deletion ctDNA molecules with a limit of detection (LOD) of ~1 and 3 fM, respectively, as well as mutant allele fractions as low as 0.0001%, during a standard acquisition time of only ~10 s per field of view. In this review, we provide a detailed roadmap for implementing iSiMREPS more broadly in research and clinical diagnostics, combining rapid analysis, high specificity, and high sensitivity.
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影响因子：
18.3
作者：
Mandal S;Li Z;Chatterjee T;Khanna K;Montoya K;Dai L;Petersen C;Li L;Tewari M;Johnson-Buck A;Walter NG
通讯作者：
Walter NG
通过阳离子共轭聚合物探测 DNA 杂交平衡，实现单核苷酸突变的高选择性检测和成像
影响因子：
7.4
作者：
Li, Zehao;Zhou, Xu;Su, Xin
通讯作者：
Su, Xin
DOI：
10.1142/s1793048013300053
发表时间：
2013-12-01
期刊：
Biophysical Reviews and Letters
影响因子：
--
作者：
Nicolai, Christopher;Sachs, Frederick
通讯作者：
Sachs, Frederick
DOI：
10.1016/j.ymeth.2018.08.002
发表时间：
2019-01-15
期刊：
Methods (San Diego, Calif.)
影响因子：
--
作者：
Johnson-Buck A;Li J;Tewari M;Walter NG
通讯作者：
Walter NG
影响因子：
11.5
作者：
Taniguchi, Kazuya;Uchida, Junji;Kato, Kikuya
通讯作者：
Kato, Kikuya