title: Detection of human immunodeficiency virus RNAs in living cells using Spinach RNA aptamers.
abstract: Many techniques currently used to measure HIV RNA production in cells suffer from limitations that include high background signal or the potential to destroy cellular context. Fluorophore-binding RNA aptamers offer the potential for visualizing RNAs directly in living cells with minimal cellular perturbation. We inserted a sequence encoding a fluorophore-binding RNA aptamer, known as Spinach, into the HIV genome such that predicted RNA secondary structures in both Spinach and HIV were preserved. Chimeric HIV-Spinach RNAs were functionally validated in vitro by testing their ability to enhance the fluorescence of a conditional fluorophore (DFHBI), which specifically binds Spinach. Fluorescence microscopy and PCR were used to verify expression of HIV-Spinach RNAs in human cells. HIV-1 gag RNA production and fluorescence were measured by qPCR and fluorometry, respectively. HIV-Spinach RNAs were fluorometrically detectable in vitro and were transcribed in human cell lines and primary cells, with both spliced and unspliced species detected by PCR. HIV-Spinach RNAs were visible by fluorescence microscopy in living cells, although signal was reproducibly weak. Cells expressing HIV-Spinach RNAs were capable of producing fluorometrically detectable virions, although detection of single viral particles was not possible. In summary, we have investigated a novel method for detecting HIV RNAs in living cells using the Spinach RNA aptamer. Despite the limitations of the present aptamer/fluorophore combination, this is the first application of this technology to an infectious disease and provides a foundation for future research into improved methods for studying HIV expression.
authors.affiliation: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Brandon D
authors.initials: BD
authors.lastname: Burch
authors.affiliation: UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Carolina
authors.initials: C
authors.lastname: Garrido
authors.affiliation: UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: dmargo@med.unc.edu.
authors.forename: David M
authors.initials: DM
authors.lastname: Margolis
mesh_terms: D052157:Aptamers, Nucleotide; D002460:Cell Line; D002478:Cells, Cultured; D006678:HIV / Q000235:genetics*; D015658:HIV Infections / Q000175:diagnosis / Q000821:virology; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D018749:RNA, Plant; D012367:RNA, Viral / Q000235:genetics*; D052156:SELEX Aptamer Technique; D013050:Spectrometry, Fluorescence / Q000379:methods; D018724:Spinacia oleracea / Q000235:genetics; D014771:Virion; D014779:Virus Replication
chemical_list: D052157:Aptamers, Nucleotide; D018749:RNA, Plant; D012367:RNA, Viral
keywords: Aptamers; HIV-RNA; Spinach