Sensing platform for nucleic-acid detection based on a 2-aminopurine probe sheared by trans-cleavage activity of the CRISPR/Cas12a system.

Target double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) can activate the trans-cleavage activity of the CRISPR/Cas12a, cutting the surrounding non-target ssDNA arbitrarily. In a typical CRISPR/Cas12a system, this non-target ssDNA, with a fluorescent tag and its quencher incorporated at both ends (ssDNA-FQ), is usually used as the reporter. Here, a 2-aminopurine probe (T-pro 4), made by inserting four 2-APs in non-target ssDNA, was screened for using as a reporter in the CRISPR/Cas12a system. Compared with ssDNA-FQ, each 2-AP probe is cleaved by the activated CRISPR/Cas12a system, multi-unit signals are generated. Therefore, the CRISPR/Cas12a system using the 2-AP probe as a reporter may be more sensitive than the CRISPR/Cas12a system which uses ssDNA-FQ as the reporter. We achieved ssDNA detection at as little as 10-11 M using the 2-AP probe as the reporter in the CRISPR/Cas12a system. Compared to the CRISPR/Cas12a system using ssDNA-FQ as the reporter, its sensitivity increased by an order of magnitude. Furthermore, the method that combines PCR and the 2-AP-probe-mediated CRISPR/Cas12a system can detect goat pox virus (GTPV) down to 8.35 × 10-2 copies per µL, 10 times lower than the method that combines PCR and the ssDNA-FQ-mediated CRISPR/Cas12a system. These results indicate that the CRISPR/Cas12a system using the screened 2-AP probe as a reporter has potential in highly sensitive detection of viruses.

Intratracheal administration of programmable DNA nanostructures combats acute lung injury by targeting microRNA-155.

Acute lung injury (ALI) is an acute inflammatory process that can result in life-threatening consequences. Programmable DNA nanostructures have emerged as excellent nanoplatforms for microRNA-based therapeutics, offering potential nanomedicines for ALI treatment. Nonetheless, the traditional systematic administration of nanomedicines is constrained by low delivery efficiency, poor pharmacokinetics, and nonspecific side effects. Here, we identify macrophage microRNA-155 as a novel therapeutic target using the magnetic bead sorting technique. We further construct a DNA nanotubular nucleic acid drug antagonizing microRNA-155 (NT-155) for ALI treatment through intratracheal administration. Flow cytometry results demonstrate that NT-155, when inhaled, is taken up much more effectively by macrophages and dendritic cells in the bronchoalveolar lavage fluid of ALI mice. Furthermore, NT-155 effectively silences the overexpressed microRNA-155 in macrophages and exerts excellent inflammation inhibition effects in vitro and ALI mouse models. Mechanistically, NT-155 suppresses microRNA-155 expression and activates its target gene SOCS1, inhibiting the p-P65 signaling pathway and suppressing proinflammatory cytokine secretion. The current study suggests that deliberately designed nucleic acid drugs are promising nanomedicines for ALI treatment and the local administration may open up new practical applications of DNA in the future.

An mTOR siRNA-Loaded Spermidine/DNA Tetrahedron Nanoplatform with a Synergistic Anti-Inflammatory Effect on Acute Lung Injury.

Acute lung injury (ALI) is characterized by severe inflammation and damage to the lung air-blood barrier, resulting in respiratory function damage and life-threatening outcomes. Macrophage polarization plays an essential role in the occurrence, development, and outcome of ALI. As drug carriers, self-assembled DNA nanostructures can potentially overcome the drawbacks and limitations of traditional anti-inflammatory agents owing to their nontoxicity, programmability, and excellent structural control at the nanoscale. A small interfering RNA (siRNA) and drug dual therapy nanoplatform are proposed and constructed here to combat ALI. The nanoplatform consists of a spermidine-assembled DNA tetrahedron and four mammalian target of rapamycin siRNAs. Spermidine serves as a mediator of drug delivery vehicle synthesis and a drug that alters macrophage polarization. Both spermidine and siRNA exert anti-inflammatory effects in vitro and in vivo by regulating the macrophage phenotype. More importantly, these factors exhibit a synergistic anti-inflammatory effect by promoting macrophage autophagy. For the first time, an anti-inflammatory dual therapy strategy that uses self-assembled DNA nanostructures as nontoxic, programmable delivery vehicles is proposed and demonstrated through this work. Future work on utilizing DNA nanostructures for the treatment of noncancerous diseases such as ALI is highly promising and desirable.