Detection of Extracellular Vesicle RNA Using Molecular Beacons

Overview

Journal iScience

Publisher Cell Press

Date 2020 Jan 21

PMID 31958756

Citations 32

Authors

Getulio Pereira de Oliveira Jr

Eric Zigon

Gaenna Rogers

Danny Davodian

Shulin Lu

Tijana Jovanovic-Talisman

Jennifer Jones

John Tigges

Sanjay Tyagi

Ionita Calin Ghiran

Affiliations

Soon will be listed here.

Abstract

Extracellular vesicles (EVs) have recently emerged as intercellular conveyors of biological information and disease biomarkers. Identification and characterization of RNA species in single EVs are currently challenging. Molecular beacons (MBs) represent an attractive means for detecting specific RNA molecules. Coupling the MBs to cell-penetrating peptides (CPPs) provides a fast, effective, and membrane-type agnostic means to deliver MBs across the plasma membrane and into the cytosol. Here, we generated RBCs-derived EVs by complement activation and tested the ability of MBs coupled with CPP to detect miRNAs from RBC-EVs. Our results showed that RBC and RBC-EVs miRNA-451a can be detected using MB-CPP, and the respective fluorescence levels can be measured by nano-flow cytometry. MB-based detection of RNA via nano-flow cytometry creates a powerful new analytical framework in which a simple addition of a reagent allows profiling of specific RNA species present within certain EV subsets.

Citing Articles

Extracellular RNA communication: A decade of NIH common fund support illuminates exRNA biology.

Amolegbe S, Johnston N, Ambrosi A, Ganguly A, Howcroft T, Kuo L J Extracell Vesicles. 2025; 14(1):e70016.

PMID: 39815775 PMC: 11735951. DOI: 10.1002/jev2.70016.

Exosomal long non-coding RNAs in lung cancer: A review.

Jiang J, Lin F, Wu W, Zhang Z, Zhang C, Qin D Medicine (Baltimore). 2024; 103(51):e38492.

PMID: 39705424 PMC: 11666185. DOI: 10.1097/MD.0000000000038492.

Emerging advanced approaches for liquid biopsy: nucleic acid assays of extracellular vesicles.

Wang D, Shen Y, Qian H, Jiang J, Xu W Theranostics. 2024; 14(19):7309-7332.

PMID: 39659566 PMC: 11626945. DOI: 10.7150/thno.102437.

Optical Imaging of Single Extracellular Vesicles: Recent Progress and Prospects.

Ma B, Li L, Bao Y, Yuan L, Liu S, Qi L Chem Biomed Imaging. 2024; 2(1):27-46.

PMID: 39473463 PMC: 11504620. DOI: 10.1021/cbmi.3c00095.

The Roles of Exosome-Derived microRNAs in Cardiac Fibrosis.

Tang X, Leng M, Tang W, Cai Z, Yang L, Wang L Molecules. 2024; 29(6).

PMID: 38542836 PMC: 10974027. DOI: 10.3390/molecules29061199.

References

Bonnet G, Tyagi S, Libchaber A, Kramer F . Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc Natl Acad Sci U S A. 1999; 96(11):6171-6. PMC: 26854. DOI: 10.1073/pnas.96.11.6171. View

Tyagi S, Kramer F . Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 1996; 14(3):303-8. DOI: 10.1038/nbt0396-303. View

Giesendorf B, Vet J, Tyagi S, Mensink E, Trijbels F, Blom H . Molecular beacons: a new approach for semiautomated mutation analysis. Clin Chem. 1998; 44(3):482-6. View

Chen M, Ma Z, Wu X, Mao S, Yang Y, Tan J . A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level. Sci Rep. 2017; 7(1):1550. PMC: 5431543. DOI: 10.1038/s41598-017-01740-1. View

Walzer K, Chi J . Trans-kingdom small RNA transfer during host-pathogen interactions: The case of P. falciparum and erythrocytes. RNA Biol. 2017; 14(4):442-449. PMC: 5411123. DOI: 10.1080/15476286.2017.1294307. View

Kuo W, Tigges J, Toxavidis V, Ghiran I . Red Blood Cells: A Source of Extracellular Vesicles. Methods Mol Biol. 2017; 1660:15-22. DOI: 10.1007/978-1-4939-7253-1_2. View

Rhee W, Jeong S . Extracellular Vesicle miRNA Detection Using Molecular Beacons. Methods Mol Biol. 2017; 1660:287-294. DOI: 10.1007/978-1-4939-7253-1_23. View

Culley S, Tosheva K, Pereira P, Henriques R . SRRF: Universal live-cell super-resolution microscopy. Int J Biochem Cell Biol. 2018; 101:74-79. PMC: 6025290. DOI: 10.1016/j.biocel.2018.05.014. View

Babatunde K, Mbagwu S, Hernandez-Castaneda M, Adapa S, Walch M, Filgueira L . Malaria infected red blood cells release small regulatory RNAs through extracellular vesicles. Sci Rep. 2018; 8(1):884. PMC: 5772623. DOI: 10.1038/s41598-018-19149-9. View

10.

LaMonte G, Philip N, Reardon J, Lacsina J, Majoros W, Chapman L . Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance. Cell Host Microbe. 2012; 12(2):187-99. PMC: 3442262. DOI: 10.1016/j.chom.2012.06.007. View

11.

Morales-Kastresana A, Musich T, Welsh J, Telford W, Demberg T, Wood J . High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer. J Extracell Vesicles. 2019; 8(1):1597603. PMC: 6586126. DOI: 10.1080/20013078.2019.1597603. View

12.

Tyagi S, Bratu D, Kramer F . Multicolor molecular beacons for allele discrimination. Nat Biotechnol. 1998; 16(1):49-53. DOI: 10.1038/nbt0198-49. View

13.

Kreimer S, Belov A, Ghiran I, Murthy S, Frank D, Ivanov A . Mass-spectrometry-based molecular characterization of extracellular vesicles: lipidomics and proteomics. J Proteome Res. 2015; 14(6):2367-84. DOI: 10.1021/pr501279t. View

14.

Nitin N, Santangelo P, Kim G, Nie S, Bao G . Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells. Nucleic Acids Res. 2004; 32(6):e58. PMC: 390380. DOI: 10.1093/nar/gnh063. View

15.

Mantel P, Hoang A, Goldowitz I, Potashnikova D, Hamza B, Vorobjev I . Malaria-infected erythrocyte-derived microvesicles mediate cellular communication within the parasite population and with the host immune system. Cell Host Microbe. 2013; 13(5):521-534. PMC: 3687518. DOI: 10.1016/j.chom.2013.04.009. View

16.

Tkach M, Thery C . Communication by Extracellular Vesicles: Where We Are and Where We Need to Go. Cell. 2016; 164(6):1226-1232. DOI: 10.1016/j.cell.2016.01.043. View

17.

Vargas D, Raj A, Marras S, Kramer F, Tyagi S . Mechanism of mRNA transport in the nucleus. Proc Natl Acad Sci U S A. 2005; 102(47):17008-13. PMC: 1287982. DOI: 10.1073/pnas.0505580102. View

18.

Craig G, Allen P, Mason M . Synthesis, characterization, and functionalization of gold nanoparticles for cancer imaging. Methods Mol Biol. 2010; 624:177-93. DOI: 10.1007/978-1-60761-609-2_12. View

19.

Illien F, Rodriguez N, Amoura M, Joliot A, Pallerla M, Cribier S . Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification. Sci Rep. 2016; 6:36938. PMC: 5107916. DOI: 10.1038/srep36938. View

20.

Mhlanga M, Tyagi S . Using tRNA-linked molecular beacons to image cytoplasmic mRNAs in live cells. Nat Protoc. 2007; 1(3):1392-8. DOI: 10.1038/nprot.2006.242. View