Nonenzymatic Autonomous Assembly of Cross-Linked Network Structures from Only Two Palindromic DNA Components for Intracellular Fluorescence Imaging of miRNAs

Abstract

The abnormal expression of miRNA-21 is often found in tumor specimens and cell lines, and thus, its specific detection is an urgent need for the diagnosis and effective therapy of cancers. In this contribution, we demonstrate a palindrome-based hybridization chain reaction (PHCR) upon the stimuli of a short oligonucleotide trigger to perform the autonomous assembly of cross-linked network structures (CNSs) for the amplification detection of miRNA-21 and sensitive fluorescence imaging of cancerous cells. The building blocks are only two palindromic hairpin-type DNA strands that are separately modified with different fluorophores (Cy3 and Cy5), which is easily combined with the catalytic hairpin assembly (CHA) technique that can further amplify the signal output. Utilizing the CHA−PHCR assay system, a small amount of miRNA-21 can activate many triggers via CHA and in turn induce the PHCR-based CNS assembly from more DNA building blocks, bringing Cy3 and Cy5 into close proximity to each other and generating ultrasensitive fluorescence resonance energy transfer signals. As a result, target miRNA can be quantitatively detected down to as low as 10 pM with high assay specificity. The coexisting nontarget miRNAs and other biomacromolecules do not interfere with signal transduction. The developed assay system is suitable for screening different expression levels of miRNA-21 in living cells by fluorescence imaging. The palindrome-based cross-linking assembly can enhance the intracellular stability of assembled nanostructures by at least fivefold and exhibit the good universality for the detection of other miRNAs. Moreover, cancerous cells can be distinguished from healthy cells, and the CHA−PHCR assay is in good accordance with the gold standard PCR method, indicating a promising platform for the diagnosis of human cancers and other genetic diseases.

Recent progress in the development of DNA-based biosensors integrated with hybridization chain reaction or catalytic hairpin assembly

“…As shown in Figure 2H , Wang et al (2018) established a general CHA-HCR sensing platform, where the outputs of CHA induced by the target miRNA could serve as the initiators for HCR circuits, showing a LOD of 2 pM. Later, Li et al (2022) integrated the concept of palindrome-based HCR (PHCR) with CHA for miR-21 detection. The presence of miR-21 catalyzed the formation of CHA products, which further initiated HCR between hairpins with palindromic domains, leading to the formation of crosslinking networks through the assembly of HCR products.…”

NIR Photocontrolled Fluorescent Nanosensor under a Six-Branched DNA Nanowheel-Induced Nucleic Acid Confinement Effect for High-Performance Bioimaging

“…In the past decade, benefiting from the favorable programmability and biocompatibility, DNA nanotechnology has become a promising choice for developing various biosensors. − Compared with colorimetric, , electrochemical, , and mass spectrometry , platforms, fluorescence imaging can intuitively exhibit real in situ photos and thus it is more conducive to conducting biosensing assays in live biosystems (e.g., cells and body). − To promote the detection sensitivity, a wealth of nucleic acid signal amplification strategies, especially the most typical protein enzyme-free catalytic reactions are proposed to confront the measurements of low-abundance targets. − Despite much effort the current DNA nanotechnology-based bioimaging methods still suffer from the following challenges.…”

Programming DNA Reaction Networks Using Allosteric DNA Hairpins

“…In recent years, research on DNA networks based on allosteric hairpin programming has grown. These networks are often based on programmable designs such as catalytic hairpin reactions (CHAs) [ 33 , 34 , 35 ] and mixed chain reactions (HCRs) [ 36 , 37 ]. These hairpin-based programmable networks show the characteristics of simple design and low background noise [ 38 ], which lead to promising application prospects.…”