Living-Cell MicroRNA Imaging with Self-Assembling Fragments of Fluorescent Protein-Mimic RNA Aptamer

Abstract

As the cellular roles of RNA abundance continue to increase, there is an urgent need for the corresponding tools to elucidate native RNA functions and dynamics, especially those of short, low-abundance RNAs in live cells. Fluorescent RNA aptamers provide a useful strategy to create the RNA tag and biosensor devices. Corn, which binds with 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO), is a good candidate for the RNA tag because of its enhanced photostability and red-shifted spectrum. Herein, we report for the first time the utilization of Corn as a split aptamer system, combined with RNA-initiated fluorescence complementation (RIFC), for monitoring RNA self-assembly and sensing microRNA. In this platform, the 28-nt Corn was divided into two nonfunctional halves (named probe I and probe II), and an additional target RNA recognition and stem part was introduced in each probe. The target RNA can trigger the self-assembly reconstitution of the Corn's G-quadruplex scaffold for DFHO binding and turn-on fluorescence. These probes can be transfected stably into mammalian cells and deliver the light-up fluorescent response to microRNA-21 (miR-21). Significantly, the probes have good photostability, with minimal fluorescence loss after continuous irradiation, and can be used for imaging of miR-21 in living mammalian cells. The proposed method is universal and could be applied to the sensing of other tumor-associated RNAs, including messenger RNA and noncoding RNA, as well as for monitoring RNA/RNA interactions. The Corn-based splitting aptamers show promising potential in the real-time visualization and mechanistic analysis of nucleic acids.

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.…”

Monitoring Clinical–Pathological Grading of Hepatocellular Carcinoma Using MicroRNA-Guided Semiconducting Polymer Dots

“…Instead, basic researchers mostly use their imaging properties under specific conditions, such as group modification and physical and chemical amendment, to expand their biological applications. As the biological value of RNA continues to increase, the field of basic research shows an urgent need for the corresponding imaging tools to elucidate RNA’s subcellular interval distribution and subtle dynamics changes, especially those of short single-stranded RNAs in live cells . However, most of these traditional RNA-tag probes are stubbornly based on fluorescence antibody immunoadsorption, fluorescein tagging, or isotopic labeling, which need complex operation steps to realize imaging; therefore, these may remain difficult to be analyzed by lagged laboratories .…”

“…As the biological value of RNA continues to increase, the field of basic research shows an urgent need for the corresponding imaging tools to elucidate RNA's subcellular interval distribution and subtle dynamics changes, especially those of short single-stranded RNAs in live cells. 27 However, most of these traditional RNA-tag probes are stubbornly based on fluorescence antibody immunoadsorption, fluorescein tagging, or isotopic labeling, which need complex operation steps to realize imaging; therefore, these may remain difficult to be analyzed by lagged laboratories. 28 In daily use, the fluorescein-labeled RNA dyes are prone to fluorescence quenching and spectral interference after dyeing due to their limitations; to some extent, the clinical application value of these dyes is limited.…”

Applications of Smartphone-Based Aptasensor for Diverse Targets Detection

“…Aptamer has been an attractive tool for various applications compared with antibodies and enzymes due to its cost-effectiveness, strong specificity, suitable pretreatment, simple operation, and short detection time [ 32 ]. Recently, aptasensors have been applied to cell imaging [ 33 , 34 ], drug delivery [ 35 , 36 ], disease treatment [ 37 , 38 ], microbial detection [ 39 , 40 ], and other fields. Hence, a smartphone-based aptasensor will be an ideal tool for POCT and analysis to obtain a fast and precise response in various fields.…”