Tharu Ram J, Hanson Emmett, Yigit Mehmet V
Department of Chemistry, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States.
The RNA Institute, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States.
ACS Chem Biol. 2025 Jul 18;20(7):1805-1812. doi: 10.1021/acschembio.5c00355. Epub 2025 Jul 9.
We report the development of a DNA hydrogel that disassembles and releases its payload in response to a target of interest. The DNA hydrogel is assembled from Y-shaped DNA motifs with polyA domains and cross-linked the small molecule cyanuric acid through hydrogen bonding. The hydrogel's structural integrity was rapidly assessed using a simple, instrumentation-free capillary migration assay that provides results within seconds. To evaluate its responsiveness to enzymatic degradation, the hydrogel was exposed to nonspecific nuclease activity using , resulting in increased mobility and decrease in fluorescence. Later, CRISPR-Cas12a was incorporated to enable programmable, target-specific hydrogel disassembly using a conserved genomic region from . Guided by crRNA sequences, the target sequences activated Cas12a to selectively degrade hydrogels. This process enabled the controlled release of various payloads, including a small-molecule drug, a fluorescent dye, a nanoparticle-based MRI contrast agent conjugated to a chemotherapeutic agent, and a model protein. To evaluate whether the hydrogel disassembly can be selectively programmed to an intended target, we tested its responsiveness against two serotypes of , , conserved genomic regions from and . To test the disassembly of this novel DNA hydrogel in the presence of a full genome, we tested the hydrogel with the genome. The target genome induced an increase in the hydrogel's mobility and loss in fluorescence with as few as 50 copies of full genome. The results demonstrate the potential of these CRISPR-responsive DNA hydrogels as intelligent platforms for target-induced imaging and therapeutic agent release, and biosensing applications.
我们报告了一种DNA水凝胶的研发情况，该水凝胶可响应感兴趣的靶标而分解并释放其负载物。这种DNA水凝胶由带有聚A结构域的Y形DNA基序组装而成，并通过氢键与小分子氰尿酸交联。使用一种简单的、无需仪器的毛细管迁移试验可在数秒内快速评估水凝胶的结构完整性。为了评估其对酶促降解的响应性，将水凝胶暴露于非特异性核酸酶活性环境中，结果导致迁移率增加和荧光降低。后来，引入了CRISPR-Cas12a，以利用来自[具体物种]的保守基因组区域实现可编程的、靶向特异性的水凝胶分解。在crRNA序列的引导下，靶序列激活Cas12a以选择性地降解水凝胶。这一过程能够可控地释放各种负载物，包括小分子药物、荧光染料、与化疗药物偶联的基于纳米颗粒的MRI造影剂以及一种模型蛋白。为了评估水凝胶分解是否可以被选择性地编程到预期靶标，我们测试了其对[具体物种]的两种血清型、来自[具体物种]的保守基因组区域以及[具体物种]的响应性。为了测试这种新型DNA水凝胶在全基因组存在情况下的分解情况，我们用水凝胶与[具体物种]基因组进行了测试。靶基因组在全基因组仅有50个拷贝时就导致水凝胶迁移率增加和荧光丧失。结果证明了这些CRISPR响应性DNA水凝胶作为用于靶标诱导成像和治疗剂释放以及生物传感应用的智能平台的潜力。
