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Control of Oncogenic miRNA Function by Light-Activated miRNA Antagomirs

  • Colleen M. Connelly
  • Alexander Deiters
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1165)

Abstract

MicroRNAs (miRNAs) are single stranded noncoding RNAs of approximately 22 nucleotides that act as posttranscriptional gene regulators by binding partially complementary sequences in the 3′ untranslated region (3′-UTR) of target messenger RNAs (mRNAs). MicroRNAs regulate many biological processes including embryonal development, differentiation, apoptosis, and proliferation and the targets of miRNAs range from signalling proteins and transcription factors to RNA binding proteins. Recently, variations in the expression of certain miRNAs have been linked to a variety of human diseases including cancer and viral infections, validating miRNAs as potential targets for drug discovery. Several tools have been developed to control the function of individual miRNAs and have been applied to study their biological role and therapeutic potential; however, common methods lack a precise level of control that allows for the study of miRNA function with high spatial and temporal resolution. Toward this goal, a light-activated miRNA antagomir for mature miR-21 was developed through the site-specific installation of caging groups on the bases of selected nucleotides. Installation of caged nucleotides led to complete inhibition of the antagomir–miRNA hybridization and inactivation of antagomir function. The miRNA-inhibitory activity of the caged antagomirs was fully restored upon decaging through a brief UV irradiation. The synthesized antagomir was applied to the photochemical regulation of miR-21 function in mammalian cells. Moreover, spatial and temporal control over antagomir activity and thus miR-21 function was obtained in mammalian cells. The presented approach enables the precise regulation of miRNA function with unprecedented spatial and temporal resolution using UV irradiation and can be readily extended to any miRNA of interest.

Key words

MicroRNA Antagomir Light-activation Caged oligonucleotides MicroRNA reporter Luciferase EGFP 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Colleen M. Connelly
    • 1
  • Alexander Deiters
    • 2
  1. 1.Department of ChemistryNorth Carolina State UniversityRaleighUSA
  2. 2.Department of ChemistryUniversity of Pittsburgh, Chevron Science CenterPittsburghUSA

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