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MnO₂ nanosheet mediated "DD-A" FRET binary probes for sensitive detection of intracellular mRNA.

ABSTRACT: The donor donor-acceptor (DD-A) FRET model has proven to have a higher FRET efficiency than donor-acceptor acceptor (D-AA), donor-acceptor (D-A), and donor donor-acceptor acceptor (DD-AA) FRET models. The in-tube and in-cell experiments clearly demonstrate that the "DD-A" FRET binary probes can indeed increase the FRET efficiency and provide higher imaging contrast, which is about one order of magnitude higher than the ordinary "D-A" model. Furthermore, MnO₂ nanosheets were employed to deliver these probes into living cells for intracellular TK1 mRNA detection because they can adsorb ssDNA probes, penetrate across the cell membrane and be reduced to Mn²⁺ ions by intracellular GSH. The results indicated that the MnO₂ nanosheet mediated "DD-A" FRET binary probes are capable of sensitive and selective sensing gene expression and chemical-stimuli changes in gene expression levels in cancer cells. We believe that the MnO₂ nanosheet mediated "DD-A" FRET binary probes have the potential as a simple but powerful tool for basic research and clinical diagnosis.

SUBMITTER: Ou M

PROVIDER: S-EPMC5297934 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundA number of RNA binding proteins (BPs) bind to A+U rich elements (AREs), commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the in vivo composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts.ResultsAll ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm (in general), or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, in vitro transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1.ConclusionsBiochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as demonstrated herein, colocalization of proteins and proteins and RNA is not always indicative of interaction. To this point, using FRET and immuno-FRET, we have demonstrated that RNA-BPs can visually colocalize without producing a FRET signal. In contrast, proteins that appear to be delimited to one or another intracellular compartment can be shown to interact when those compartments are juxtaposed.

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MnO₂ nanosheet mediated "DD-A" FRET binary probes for sensitive detection of intracellular mRNA.

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MnO₂ nanosheet mediated "DD-A" FRET binary probes for sensitive detection of intracellular mRNA.