Engineering of a palindrome-crosslinked DNA nanoaggregate for rapid detection of circular RNA and precise identification of lung cancer

Abstract

Circular RNAs (circRNAs) are endogenous covalently closed non-coding RNAs with cell-/developmental-stage-/tissue-specific expression patterns, and they can act as the miRNA sponges and gene transcription regulatory factors to influence numerous biological processes. Herein, we develop a palindrome-crosslinked DNA nanoaggregate system to rapidly detect circRNA and precisely identify lung cancer. We utilize a self-assembled palindromic DNA nanosphere (DS) as the spatial-confinement scaffold to anchor hairpin probes (HP) for the formation of the hybrid assemblies (DSH). The presence of target circSATB2 can hybridize with the hairpin probe to expose the locked palindromic sequence, initiating the cross-linking of the palindromic ends to form a self-catenated structure through intermolecular hybridization. Then the hybridized palindromic ends serve as the self-primers to initiate extension reaction and eventually assemble into the net-like crosslinked DNA nanoaggregates, resulting in the recovery of Cy5 signals. Taking advantage of the excellent antidegradation capability and superior kinetic behavior of DSH nanostructure, high amplification efficiency of Klenow Fragment polymerase (KF)-mediated extension reaction, and signal enhancement induced by the DNA nanoaggregates, this nanosystem enables mix-and-read detection of circSATB2 within 30 min under isothermal conditions (37 °C) with a limit detection of 77.56 fM. Moreover, it is capable of measuring intracellular circSATB2 with single-cell sensitivity, exploring its biological functions, and precisely identifying different stages (I/II/III) and subtypes (IA1/IA2/IA3/IB) of lung cancers, holding great potential in early screening of lung cancers.