Self-assembled palindromic DNA nanostructure for orthogonal entropy-driven amplification and multiplexed fluorescence fingerprinting of intracellular RNAs.

Simultaneous imaging of multiple intracellular RNA biomarkers is essential for precise molecular subtyping of cancers, but achieving orthogonal signal amplification with a low background remains a challenge. Herein, we design an entropy-driven palindromic DNA nanostructure by integrating four orthogonal catalytic circuits into a single nanostructure for accurate discrimination of different breast cancer cell subtypes based on multiplexed imaging of miRNA-21, miRNA-373, miRNA-155, and MnSOD mRNA. This system is constructed from two cruciform modules. Cruciform module 1 is formed by four substrate complexes with each consisting of a quencher-labeled template, a fluorophore-labeled reporter (Alexa Fluor 405, 488, 568, and 647) and an assistant strand carrying a palindromic arm for co-localizing the fluorophores and quenchers to suppress fluorescence. Cruciform module 2 is assembled from four fuel strands sharing the same palindromic sequence. Hybridization of two modules via their complementary palindromic arms generates self-assembled palindromic DNA nanostructure (SAPDN). Upon cellular internalization of SAPDN, each target RNA binds a specific toehold on its corresponding substrate complex to trigger an entropy-driven strand-displacement cascade, releasing a target-probe intermediate. Subsequent invasion by the fuel strand results in the liberation of intact target, the displacement of reporter strand, and the separation of fluorophore from the quencher to restore fluorescence. The liberated RNA targets can activate new substrate complexes to achieve catalytic signal amplification. By mapping the four fluorescence signatures inside living cells, the SAPDN platform can effectively differentiate different breast cancer subtypes, holding great promise in advanced molecular diagnostics.