Shattering kinetic constraints: hierarchically activatable DNAzyme nanoantennas for in situ mRNA imaging and precise cancer theranostics

Abstract

Cancer progressions are complicated events with involvement of abnormal expression of different RNAs. DNAzyme-based imaging-guided gene therapy offers promising prospects for effectively treating serious diseases, but it is constrained by unconditional activation, inefficient delivery, and limited amplification capacity. Herein, we construct shattering kinetic constraints: hierarchically activatable DNAzyme nanoantennas for in situ mRNA imaging and precise cancer theranostics. This nanosystem incorporates multifunctional modules including an aptamer-mediated tumor-target module, an i-motif-gated recognition module, a metal ion-activated amplification module, and an antisense oligonucleotide (ASO)-induced therapy module. Once encountering cancer cells, hairpin 1 (H1) recognizes the nucleolin via an aptamer AS1411, and meanwhile the i-motif in H1 undergoes structure switching at extracellular acid pH, enabling the cross-opening of H2, H3, H4, H5 for in situ self-assembly of two-sided DNAzyme nanoantennas. Upon internalization by cancer cells, DNAzymes in the nanoantenna cleave the substrates with intracellular Mg²⁺ as a cofactor, releasing abundant molecular beacons containing ASOs. ASOs hybridize with Egr-1 mRNAs, inducing the recovery of Cy5 fluorescence for Egr-1 mRNA imaging and silencing. This nanoantenna achieves a limit of detection of 7.46 fM for in vitro assay. It can profile Egr-1 mRNA level in diverse human cells, and distinguish Egr-1 mRNA level in breast cancer tissues and healthy counterparts. Moreover, it can be applied for high-contrast imaging of Egr-1 mRNA in vivo, efficient gene silencing, and enhanced tumor ablation, with promising applications in smart gene therapeutics and precise nanomedicines.