Biology Reference
In-Depth Information
Chapter 8
A Fluorescence Correlation Spectroscopy-Based Enzyme
Assay for Human Dicer
Eileen Magbanua and Ulrich Hahn
Abstract
We used fluorescence correlation spectroscopy (FCS) to establish an in vitro assay to investigate RNase
activity of human Dicer (Werner et al., Biol Chem 393(3):187-193). FCS allows investigating the interac-
tions of different particles due to their differing diffusion mobility, provided that one of the interacting
partners exhibit a fluorescence label. In our case we used a fluorophore-labeled double-stranded RNA
(dsRNA) as substrate to monitor Dicer activity. The dsRNA was cleaved by the enzyme resulting in a five-
nucleotide-short single-stranded RNA (ssRNA) fragment carrying the fluorophore, which could be distin-
guished from the substrate and unlabeled second product by FCS. Furthermore, we refer to additional
(control) experiments to confirm obtained data.
Key words
FCS, Human Dicer, Dicer assay, RNase III, dsRNA
1
Introduction
Fluorescence correlation spectroscopy (FCS) is a method, which
enables analyzing fluctuations of diffusing fluorescent molecules.
Different fluorescent molecules which vary in mass by a factor of
eight can be distinguished from each other [
1
]. Fulfilling this
requirement FCS can easily be used to investigate interactions of
fluorescently labeled particles and for example in vitro enzyme
activity assays [
2
-
4
]. Further advantages of FCS are the small sam-
ple amounts as well as a small detection volume of approximately
0.1 fl, which represents less than the volume of a standard bacterial
cell. Here we used FCS to investigate the RNase activity of human
Dicer [
5
]. Dicer plays a key role during gene regulation by RNA
interference (RNAi). It is responsible for the delivery of substrate
for RISC by cleaving double-stranded RNA (dsRNA) to small
interfering RNAs (siRNA) or microRNAs (miRNAs) which are
than interacting with further proteins to target selected mRNAs
resulting in gene silencing [
6
-
8
].
