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Methods Mol Biol. 2018;1680:101-121. doi: 10.1007/978-1-4939-7339-2_7.

Kinetic Analysis of Small Silencing RNA Production by Human and Drosophila Dicer Enzymes In Vitro.

Author information

1
Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, 521A Physiology Building, Baltimore, MD, 21205, USA.
2
Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, 521A Physiology Building, Baltimore, MD, 21205, USA. fukunaga@jhmi.edu.

Abstract

Dicer enzymes produce small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), which then are loaded into Argonaute proteins and act as sequence-specific guides. A powerful tool to understand the molecular mechanism of small silencing RNA production by Dicers is an in vitro RNA processing assay using recombinant Dicer proteins. Such biochemical analyses have elucidated the substrate specificities and kinetics of Dicers, the mechanism by which the length of small RNAs produced by Dicers is determined, and the effects of Dicer-partner proteins and endogenous small molecules such as ATP and inorganic phosphate on small RNA production by Dicers, among others. Here, we describe methods for in vitro small RNA production assay using recombinant human and Drosophila Dicer proteins.

KEYWORDS:

Dicer; Kinetics; RNA silencing; miRNA; siRNA

PMID:
29030844
DOI:
10.1007/978-1-4939-7339-2_7
[Indexed for MEDLINE]

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