Rapid kinetic fingerprinting of single nucleic acid molecules by a FRET-based dynamic nanosensor.
Rapid kinetic fingerprinting of single nucleic acid molecules by a FRET-based dynamic nanosensor.
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DOI：
10.1016/j.bios.2021.113433
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发表时间：
2021-10-15
影响因子：
12.6
通讯作者：
Walter NG
中科院分区：
文献类型：
--
作者：
Khanna K;Mandal S;Blanchard AT;Tewari M;Johnson-Buck A;Walter NG
生物流体来源的无细胞核酸如microRNA（miRNA）和循环肿瘤来源的DNA（ctDNA）已经成为有前途的疾病生物标志物。通过数字PCR和下一代测序对这些生物标志物进行常规检测，尽管灵敏度很高，但需要耗时的提取和扩增步骤，这也增加了样本丢失和交叉污染的风险。为了实现直接、快速和无扩增检测miRNA和ctDNA，具有近乎完美的特异性和单分子水平的灵敏度，我们在此设计了单分子动力学指纹分析，称为通过平衡泊松采样的分子内单分子识别（iSiMREPS）。iSiMREPS利用了一种动态DNA纳米传感器，包括一个表面锚和一对荧光检测探针：一个探针将目标分子捕获到表面上，而另一个探针通过分子内单分子Förster共振能量转移（smFRET）瞬时询问目标以生成动力学指纹，该指纹由单分子荧光显微镜记录，并在动力学过滤和数据分析后识别目标。我们优化了传感器的设计，使用甲酰胺，以进一步加速指纹动力学，并通过使用支点介导的链置换，以减少背景去除非目标结合的探针，以最大限度地提高灵敏度。我们发现，iSiMREPS可以在短短10秒内检测到两种不同的、有希望的癌症生物标志物-miR-141和一种常见的EGFR外显子19缺失-达到约3 fM的检测限（LOD），过量野生型中的突变等位基因比例低至百万分之一，即0.0001%。我们预计，iSiMREPS将在研究和临床诊断的基础上，其快速检测，高特异性，灵敏度和普适性的功能实用。
Biofluid-derived cell-free nucleic acids such as microRNAs (miRNAs) and circulating tumor-derived DNAs (ctDNAs) have emerged as promising disease biomarkers. Conventional detection of these biomarkers by digital PCR and next generation sequencing, although highly sensitive, requires time-consuming extraction and amplification steps that also increase the risk of sample loss and cross-contamination. To achieve the direct, rapid, and amplification-free detection of miRNAs and ctDNAs with near-perfect specificity and single-molecule level sensitivity, we herein designed a single-molecule kinetic fingerprinting assay, termed intramolecular single-molecule recognition through equilibrium Poisson sampling (iSiMREPS). iSiMREPS exploits a dynamic DNA nanosensor comprising a surface anchor and a pair of fluorescent detection probes: one probe captures a target molecule onto the surface, while the other transiently interrogates the target to generate kinetic fingerprints by intramolecular single-molecule Förster resonance energy transfer (smFRET) that are recorded by single-molecule fluorescence microscopy and identify the target after kinetic filtering and data analysis. We optimize the sensor design, use formamide to further accelerate the fingerprinting kinetics, and maximize sensitivity by removing non-target-bound probes using toehold-mediated strand displacement to reduce background. We show that iSiMREPS can detect, in as little as 10 seconds, two distinct, promising cancer biomarkers—miR-141 and a common EGFR exon 19 deletion—reaching a limit of detection (LOD) of ~3 fM and a mutant allele fraction among excess wild-type as low as 1 in 1 million, or 0.0001%. We anticipate that iSiMREPS will find utility in research and clinical diagnostics based on its features of rapid detection, high specificity, sensitivity, and generalizability.
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影响因子：
4.6
作者：
Dunn KE;Trefzer MA;Johnson S;Tyrrell AM
通讯作者：
Tyrrell AM
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：
10.1016/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
DOI：
10.1073/pnas.1208715109
发表时间：
2012-09-04
影响因子：
11.1
作者：
Schmitt, Michael W.;Kennedy, Scott R.;Loeb, Lawrence A.
通讯作者：
Loeb, Lawrence A.
影响因子：
8.8
作者：
Bryant, R. J.;Pawlowski, T.;Catto, J. W. F.;Marsden, G.;Vessella, R. L.;Rhees, B.;Kuslich, C.;Visakorpi, T.;Hamdy, F. C.
通讯作者：
Hamdy, F. C.