Nanoamplicon Comparator for Live-Cell MicroRNA Imaging

Abstract

As an investigative tool, live-cell imaging requires superior probe design to guarantee imaging quality and data validity. The ability to simultaneously address the robustness, sensitivity, and consistency issues in a single-assay system is highly desired, but it remains a largely unsolved challenge. We describe herein a probe-design strategy called a nanoamplicon comparator (NAC) and demonstrate its proofof-concept utility in intracellular microRNA (miRNA) imaging. This novel designer architecture builds upon spherical nucleic acids (SNAs) for robustness, catalytic hairpin assembly (CHA) for sensitivity, and upconversion nanoparticles (UNPs) for consistency. A catalytic circuit comprising a UNP−hairpin-DNA (UNP-HDNA) conjugate and a hairpin-DNA−organic-fluorophore (HDNA-F) conjugate as probe responds to target miRNA and generates the UNP-HDNA−HDNA-F complex as an NAC for quantitative UNP-to-organic-fluorophoreluminescence-resonance-energy-transfer (LRET) imaging against a native UNP-emission reference channel. An imaging application with miR21 shows the ability to monitor miRNA-expression levels across different cell lines and under an external stimulus.

A photo zipper locked DNA nanomachine with an internal standard for precise miRNA imaging in living cells

“…2e ), which is much improved compared with many previous reported UCNP-based miRNA detection probes. 50 The introduction of the DNA nanomachine on the UCNP surface contributed to impressive enhancement of the detection sensitivity. 23 , 51 , 52 To further demonstrate the amplification effect of the PZ-DNA nanomachine, a FAM labelled P-DNA walker was prepared by conjugating the dye FAM to the photo zipper, and immobilized on the UCNP surface with S-DNA at a molar ratio of 1 : 10 to prepare a PF-DNA nanomachine (Fig.…”

Spherical nucleic acids: Organized nucleotide aggregates as versatile nanomedicine

“…[ 146 ] Nanoamplicon comparator probe has the ability to monitor miRNA expression levels in different cell lines under external stimuli. [ 142 ]…”

Label- and enzyme-free plasmon-enhanced single molecule fluorescence detection of HIV DNA fragments based on a catalytic hairpin assembly

“…23 Compared with the hybridization chain reaction, 24 it takes less time to achieve a high level signal amplification. However, many target-triggered CHA fluorescence methods demand labeling of the hairpin probes, 25,26 and fluorescent labeling introduces additional complexity and cost, which may limit their actual applicabilities. Therefore, it is essential to propose a convenient and cheap fluorescence signal reading method with a fast and simple operation, which can detect the target DNA sensitively and selectively.…”