Anal Chem. 2023 Sep 19;95(37):13796-13803. doi: 10.1021/acs.analchem.3c01064. Epub 2023 Aug 31.
Double-stranded (ds) oligonucleotide probes composed of quencher-dye sequence pairs outperform analogous single-stranded (ss) probes due to their superior target sequence specificity without any prerequisite target labeling. Optimizing sequence combinations for dsprobe design requires promoting a fast, accurate response to a specific target sequence while minimizing spontaneous dsprobe dissociation events. Here, flow cytometry is used to rapidly interrogate the stability and selective responsiveness of 20 candidate LNA and DNA dsprobes to a 24 base-long segment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and ∼243 degenerate RNA sequences serving as model variants. Importantly, in contrast to quantifying binding events of dye-labeled targets via flow cytometry, the current work employs the Förster resonance energy transfer (FRET)-based detection of unlabeled RNA targets. One DNA dsprobe with a 15-base-long hybridization partner containing a central abasic site emerged as very stable yet responsive only to the SARS-CoV-2 RNA segment. Separate displacement experiments, however, indicated that ∼12% of these quencher-capped hybridization partners remain bound, even in the presence of an excess SARS-CoV-2 RNA target. To examine their quenching range, additional titration studies varied the ratios and spatial placement of nonquencher and quencher-capped hybridization partners in the dsprobes. These titration studies indicate that these residual, bound quencher-capped partners, even at low percentages, act as nodes, enabling both static quenching effects within each residual dsprobe as well as longer-range quenching effects on neighboring FAM moieties. Overall, these studies provide insight into practical implications for rapid dsprobe screening and target detection by combining flow cytometry with FRET-based detection.
双链(ds)寡核苷酸探针由淬灭剂-染料序列对组成,由于其具有优越的靶序列特异性,而无需任何前提靶标标记,因此优于类似的单链(ss)探针。优化 dsprobe 设计的序列组合需要促进对特定靶序列的快速、准确响应,同时最小化自发的 dsprobe 解离事件。在这里,流式细胞术用于快速检测 20 种候选 LNA 和 DNA dsprobe 对 24 个碱基长的严重急性呼吸综合征冠状病毒 2(SARS-CoV-2)RNA 片段和作为模型变体的约 243 个简并 RNA 序列的稳定性和选择性响应。重要的是,与通过流式细胞术定量染料标记靶标结合事件不同,目前的工作采用基于Förster 共振能量转移(FRET)的未标记 RNA 靶标的检测。一种具有包含中央无碱基位点的 15 个碱基长杂交伴侣的 DNA dsprobe 表现出非常稳定但仅对 SARS-CoV-2 RNA 片段有响应。然而,单独的置换实验表明,即使存在过量的 SARS-CoV-2 RNA 靶标,这些淬灭帽杂交伴侣中仍有约 12%保持结合。为了检查它们的猝灭范围,额外的滴定研究改变了 dsprobe 中无猝灭剂和淬灭帽杂交伴侣的比例和空间放置。这些滴定研究表明,这些残留的、结合的淬灭帽杂交伴侣,即使在低百分比的情况下,也作为节点,使每个残留的 dsprobe 内的静态猝灭效应以及对相邻 FAM 部分的长程猝灭效应成为可能。总体而言,这些研究通过将流式细胞术与基于 FRET 的检测相结合,为快速 dsprobe 筛选和靶标检测提供了实际意义。