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Probing cytoplasmic and nuclear microRNAs in single living cells via plasmonic affinity sandwich assay.

ABSTRACT: MicroRNAs (miRNAs) regulate the expression of mRNAs in cells. The determination of subcellular miRNAs in single living cells is essential for understanding the subcellular localizations and functions of miRNAs as well as the microheterogeneity of cells. However, current approaches fail either to keep the cells alive or maintain their original cellular composition or are unable to provide subcellular resolution. Herein, we presented a new approach, called plasmonic affinity sandwich assay (PASA), for rapid and ultrasensitive probing of trace cytoplasmic and nuclear miRNAs in single living cells. It combined efficient in vivo subcellular extraction with ultrasensitive plasmon-enhanced Raman scattering (PERS) detection. By virtue of a micromanipulator, target miRNAs in the cytoplasm and nucleus were first specifically extracted from single living cells by gold thinlayer-coated glass microprobes modified with a half complementary sequence to the target miRNA and then, they were labelled with silver nanotags modified with a Raman reporter and the other half complement. Sandwich-like complexes of extraction sequence-target miRNA-labelling sequence were formed on the extraction microprobe, which were subjected to PERS detection. The subcellular resolution of this approach was confirmed with miR-29b (predominantly localized in the nuclei) and miR-29a (mainly located in the cytoplasm), whereas the quantitative capability was verified with three cytoplasmic miRNAs including miR-21, miR-155 and miR-203. This approach obviated tedious steps such as subcellular fractionation and enzymatic amplification, and it required only 10 min. It could be a promising tool to provide insights into subcellular localizations, functions and microheterogeneity of miRNAs.

SUBMITTER: Liu J

PROVIDER: S-EPMC6148463 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Probing cytoplasmic and nuclear microRNAs in single living cells <i>via</i> plasmonic affinity sandwich assay.

Liu Jia J Wen Yanrong Y He Hui H Chen Hong-Yuan HY Liu Zhen Z

Chemical science 20180725 36

MicroRNAs (miRNAs) regulate the expression of mRNAs in cells. The determination of subcellular miRNAs in single living cells is essential for understanding the subcellular localizations and functions of miRNAs as well as the microheterogeneity of cells. However, current approaches fail either to keep the cells alive or maintain their original cellular composition or are unable to provide subcellular resolution. Herein, we presented a new approach, called plasmonic affinity sandwich assay (PASA), ...[more]

PMID: 30288244

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Project description:BackgroundAntigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism.Methods and findingsIn parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections.ConclusionsThe C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent driven antigen sandwich assays for the Ebolavirus genus.

Dataset Information

Probing cytoplasmic and nuclear microRNAs in single living cells via plasmonic affinity sandwich assay.

Publications

Probing cytoplasmic and nuclear microRNAs in single living cells <i>via</i> plasmonic affinity sandwich assay.

Similar Datasets

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