miRNA inhibition by proximity-enabled Dicer inactivation

Abstract

microRNAs (miRNAs) are considered as master regulators of biological processes. Dysregulation of miRNA expression has been implicated in many human diseases. Driven by the key biological roles and the therapeutic potential, developing methods for miRNA regulation has become an intense research area. Due to favorable pharmacological properties, small molecule-based miRNA inhibition emerges as a promising strategy and significant progresses have been made. However, it remains challenging to regulate miRNA using small molecules because of the inherent difficulty in RNA targeting and inhibition. Herein we outline the workflow of generating bifunctional small molecule inhibitors blocking miRNA biogenesis through proximity-enabled inactivation of Dicer, an enzyme required for the processing of precursor miRNA (pre-miRNA) into mature miRNA. By conjugating a weak Dicer inhibitor with a pre-miRNA binder, the inhibitor can be delivered to the Dicer processing site associated with the targeted pre-miRNA, and as a result inhibiting Dicer-mediated pre-miRNA processing. This protocol can be applicable in producing bifunctional inhibitors for different miRNAs.

Keywords: Dicer; RNA binder; RNase inhibitor; miRNA inhibition.

Fig. 1.

An illustration of the approach: (a) miRNA is produced from pre-miRNA by Dicer processing; (b) Weak Dicer inhibitor does not affect the activity of Dicer; (c) Bifunctional molecule inhibits the enzymatic activity of Dicer complexed with pre-miRNA.

Fig. 2.

An illustration of the FP-based screening and binding assay. Upon excitation by polarized light, (a) a free fluorescent pre-miRNA binder emits depolarized light; (b) when complexed to pre-miRNA, it gives polarized light due to reduced tumbling; (c) a strong pre-miRNA binder replaces the fluorescent binder from the complex, resulting in decreased polarity of the light emitted.

Fig. 3.

The structures of kanamycin and its fluorophore-tagged derivatives.

Fig. 4.

Structures of Neomycin and its derivatives.

Fig. 5.

Fluorescence polarization of NF in presence of pre-miR-21.

Fig. 6.

Structures of Dicer inhibitors.

Fig. 7.

Synthesis of the bifunctional conjugates via click chemistry.

Fig. 8.

Electrophoresis gel analysis of ³²P labeled pre-miR-21 cleavage mediated by Dicer in the presence of tested compounds.

Fig. 9.

RT-qPCR analysis of miRNA level in HEK293T cells.