Spatiotemporal control of microRNA function using light-activated antagomirs

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional gene regulators and have been shown to regulate many biological processes including embryonal development, cell differentiation, apoptosis, and proliferation. Variations in the expression of certain miRNAs have been linked to a wide range of human diseases - especially cancer - and the diversity of miRNA targets suggests that they are involved in various cellular networks. Several tools have been developed to control the function of individual miRNAs and have been applied to study their biogenesis, 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. Light-activated miRNA antagomirs for mature miR-122 and miR-21 were 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 thus inactivation of antagomir function. The miRNA-inhibitory activity of the caged antagomirs was fully restored upon decaging through a brief UV irradiation. The synthesized antagomirs were applied to the photochemical regulation of miRNA function in mammalian cells. Moreover, spatial control over antagomir activity was obtained in mammalian cells through localized UV exposure. The presented approach enables the precise regulation of miRNA function and miRNA networks with unprecedented spatial and temporal resolution using UV irradiation and can be extended to any miRNA of interest.

Figure 1

Light-activation of miR-122 inhibition and subsequent luciferase expression by decaging of a miR-122 antagomir in Huh7 cells. Cells were transfected with the caged and non-caged miR-122 antagomir, and UV irradiation (365 nm, 25 W) for 5 min efficiently activated antagomir function. The error bars represent standard deviations from three independent experiments.

Figure 2

Light-activation of miR-21 inhibition and subsequent luciferase expression by decaging of a miR-21 antagomir in Huh7 cells. Cells were transfected with the caged and non-caged miR-21 antagomir, and UV irradiation (365 nm, 25 W) for 5 min efficiently activated antagomir function. The error bars represent standard deviations from three independent experiments.

Figure 3

Temporal control over miR-21 inhibition and subsequent luciferase expression by decaging of a miR-21 antagomir in Huh7 cells. Cells were transfected with the caged miR-21 antagomir, and were irradiated (365 nm, 5 min) at 0, 12, 24, 36, 42, and 46 hours post transfection. The error bars represent standard deviations from three independent experiments.

Figure 4

Light-activation of EGFP expression by decaging of a miR-21 antagomir in Huh7 cells transiently transfected with an EFGP sensor for miR-21 function. UV irradiation (365 nm, 5 min) restored antagomir activity and inhibited miR-21 function. The miR-122 antagomir was used as a negative control since it shows no crossreactivity with the reporter.

Figure 5

Spatial activation of EGFP expression by decaging of a miR-21 antagomir in a localized fashion. Huh7 cells were co-transfected with an EGFP sensor for miR-21 function and the caged miR-21 antagomir (100 pmol). Cells were irradiated at 365 nm using a LED fiber optics probe (Prizmatix) and were imaged after 48 hrs on a Zeiss Axio Observer inverted microscope (10× objective, 2×2 tile scan). The EGFP channel and corresponding bright field images are shown.

Scheme 1

Regulation of gene expression through photocontrol of endogenous miRNAs with nucleobase-caged antagomirs.

Scheme 2

Synthesis of the NPOM-caged 2′OMe U phosphoramidite 7.