A key challenge for the discrete distribution-based Förster resonance energy transfer system (D-FRET) is the reduced intensity and stability of signal probes in complex biological matrices. Here, we present a spatially confined FRET (SC-FRET) probe with a stable structure and strong signal output. It consists of multivalent FRET pairs labeled with FAM or TAMRA. In this assay, p53 DNA was chosen as a model hairpin probe (HP), and two kinds of branched DNA probes (ssDNA-FAM, ssDNA-TAMRA) were involved. Under the action of p53 DNA, the unfolded HP acts as a primer to initiate polymerization extension of KFP polymerase and cleavage of Nb.BbvCI endonuclease, which produces plenty of ssDNA (primer-DNA). The branched DNA is designed to have the same binding core and different sticky ends, the core part of which can self-assemble to form X-shaped branched DNA (X-FAM or X-TAMRA), and the sticky ends of which are complementary to the primer-DNA. Therefore, the primer-DNAs released during the polymerization cleavage process will combine a large number of X-FAM and X-TAMRA in a limited space through complementary base pairing. Fluorescence was transferred from FAM to TAMRA, and a strong FRET response was generated by the locational effects. The proposed SC-FRET system based on the multivalent assembly of branched DNA exhibited a strong FRET response with an LOD of 0.01394 pM. Importantly, it also showed a high-contrast and stable FRET response in HeLa cells. Its superior biological stability is attributed to the large steric hindrance of the compact and rigid frame of the SC-FRET probe, which helps prevent intracellular degradation and provides a powerful tool for biomedical research.

中文翻译：

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基于空间受限荧光共振能量转移和分支 DNA 多价组装的 p53 DNA 灵敏检测

基于离散分布的 Förster 共振能量转移系统 (D-FRET) 的一个关键挑战是复杂生物基质中信号探针的强度和稳定性降低。在这里，我们提出了一种具有稳定结构和强信号输出的空间受限 FRET (SC-FRET) 探针。它由标有 FAM 或 TAMRA 的多价 FRET 对组成。本实验选择p53 DNA作为模型发夹探针（HP），涉及两种分支DNA探针（ssDNA-FAM、ssDNA-TAMRA）。在 p53 DNA 的作用下，未折叠的 HP 作为引物启动 KFP 聚合酶的聚合延伸和 Nb.BbvCI 核酸内切酶的裂解，产生大量 ssDNA（引物-DNA）。分支的 DNA 被设计成具有相同的结合核心和不同的粘性末端，其核心部分可自组装形成X形分支DNA（X-FAM或X-TAMRA），其粘性末端与引物-DNA互补。因此，在聚合裂解过程中释放的引物-DNA 将通过互补碱基配对在有限空间内结合大量 X-FAM 和 X-TAMRA。荧光从 FAM 转移到 TAMRA，位置效应产生了强烈的 FRET 响应。提出的基于分支 DNA 多价组装的 SC-FRET 系统表现出强烈的 FRET 响应，LOD 为 0.01394 pM。重要的是，它还在 HeLa 细胞中显示出高对比度和稳定的 FRET 反应。其卓越的生物稳定性归因于 SC-FRET 探针紧凑而刚性框架的大空间位阻，