Detection of human immunodeficiency virus RNAs in living cells using Spinach RNA aptamers

Brandon D. Burch, Carolina Garrido, David M. Margolis

Research output: Research - peer-reviewArticle

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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.

LanguageEnglish (US)
Pages141-146
Number of pages6
JournalVirus Research
Volume228
DOIs
StatePublished - Jan 15 2017

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Nucleotide Aptamers
Spinacia oleracea
HIV
RNA
Fluorescence Microscopy
Virion
Fluorescence
Polymerase Chain Reaction
In Vitro Techniques
Fluorometry
Communicable Diseases
HIV-1
Genome
Technology
Cell Line

Keywords

  • Aptamers
  • HIV-RNA
  • Spinach

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases
  • Cancer Research

Cite this

Detection of human immunodeficiency virus RNAs in living cells using Spinach RNA aptamers. / Burch, Brandon D.; Garrido, Carolina; Margolis, David M.

In: Virus Research, Vol. 228, 15.01.2017, p. 141-146.

Research output: Research - peer-reviewArticle

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