终端脱氧核苷酸转移酶（TdT）在临床医学和血液病理学中的生理功能分析突出了其作为白血病诊断的诊断生物标志物的重要性。因此，利用三维（3D）DNA纳米结构的空间限制效应，我们报道了一种基于目标触发的分子信标（MB）组装的快速实时TdT活性分析方法。在这种策略中，通过交联网络杂交链反应（HCR），首先通过工程化的方法构建了3D的DNA纳米结构。然后，设计了一系列带有中间聚胸腺嘧啶（poly-T）环的MB，将其与支架DNA纳米结构连接。从而使得获得的MB-DNA纳米结构体内或体外含有大量自由的3’-末端羟基（OH）。此外，不同MB之间的距离变近，MB的局部浓度得到显著提高，这是由于MB在这个DNA纳米结构上的限制效应。一旦遇到目标TdT，自由的-OH基团可以立即被TdT识别并催化无模板的腺嘌呤核苷酸的无序合成，导致多个多聚A链的产生，这些链能够通过分子内加速组装过程与许多MB发生快速反应。从MB发出的荧光在时间上得到了显著增强，从而可用于稳定分析TdT。我们的观察结果表明，基于DNA纳米结构的空间限制效应能够增加分子碰撞频率，加速反应动力学，而超级DNA纳米结构表现出更好的核酸酶抵抗性，以维持信号的稳定性。凭借这些优势，可以高灵敏度、高特异性和生物稳定性快速检测TdT。

Physiological function analysis of terminal deoxynucleotidyl transferase (TdT) in clinical medicine and hematopathology highlights its significance to be extensively utilized as a diagnostic biomarker for leukemia diagnosis. Herein, taking advantage of the spatial-confinement effect on a three-dimensional (3D) DNA nanoarchitecture, we reported a target-triggered intramolecular accelerated molecular beacon (MB) assembly for rapid and real-time analysis of TdT activity. In this strategy, the 3D DNA nanoarchitecture is first engineered via a cross-linking network hybridization chain reaction (HCR). A number of MBs, which were designed with a polythymine (poly-T) loop, were then conjugated on the scaffold DNA nanoarchitecture, allowing the obtained MB-DNA nanoarchitecture to contain lots of free 3'-hydroxyl (OH) termini inside or outside the super DNA nanostructure. Moreover, the distance between different MBs is closed, and the local concentration of MB is significantly improved owing to the confinement of MBs on this DNA nanoarchitecture. Once encountered with target TdT, the free -OH groups can be recognized by TdT immediately to catalyze the template-independent incorporation of adenine nucleotides, which results in the generation of multiple poly-A chains that rapidly react with many MBs via an intramolecular accelerated assembly process. The time-dependent substantial enhancement of the fluorescence from MBs can thus be applied for robustly analyzing TdT. Our observations suggest that the DNA nanostructure-based spatial confinement effect enables a high molecular collision frequency to accelerate the reaction kinetics, and the super DNA nanoarchitecture exhibits a better nuclease resistance to maintain signal stability. With these advantages, TdT can be rapidly detected with high sensitivity, specificity, and biostability.