下一代原位杂交链式反应：更高的增益、更低的成本、更高的耐用性,ACS Nano

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下一代原位杂交链式反应：更高的增益、更低的成本、更高的耐用性
ACS Nano ( IF 15.8 ) Pub Date : 2014-04-08 00:00:00 , DOI: 10.1021/nn405717p
Harry M T Choi ₁ , Victor A Beck , Niles A Pierce

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杂交链式反应 (HCR) 在不同的环境下提供多重、等温、无酶的分子信号放大。在信号与背景非常重要的完整脊椎动物胚胎中，HCR原位扩增能够同时绘制多个目标 mRNA，解决了生物科学中长期存在的挑战。通过这种方法，与 mRNA 靶标互补的 RNA 探针会触发链式反应，其中亚稳态荧光团标记的 RNA 发夹自组装成束缚的荧光放大聚合物。HCR 的特性导致在固定的整装斑马鱼胚胎（脊椎动物发育研究的标准模型系统）内实现简单的多重分析、深样本穿透、高信号背景和清晰的亚细胞信号定位。然而，RNA 试剂价格昂贵且容易被酶降解。此外，用于破坏非特异性发夹结合稳定的严格杂交条件也会降低 HCR 聚合的能量驱动力，从而在背景最小化和信号最大化之间建立权衡。在这里，我们通过证明使用允许的原位扩增条件（0% 甲酰胺，室温）可以实现低背景水平来消除这种权衡，并设计下一代 DNA HCR 放大器，最大限度地提高每个聚合步骤的自由能效益，同时保留条件聚合基础的动力学捕获特性，可显着提高信号增益、降低试剂成本并提高试剂耐久性。

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Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability

Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability.

更新日期：2014-04-08

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