A Fluorescence Correlation Spectroscopy-Based Enzyme Assay for Human Dicer - miRNA Maturation: Methods and Protocols - page 100

Dicer activity and reduces the risk of artifacts due to unspecific
interactions or photodynamic processes. As Dicer activity is strongly
dependent on the presence of Mg 2+ -ions for instance replacement
of Mg 2+ - by Ca 2+ -ions leads to a significant inhibition of Dicer
activity [ 11 ], and thus diffusion time should remain constant due
to remaining uncleaved substrate even after enzyme addition.
Furthermore, Dicer does solely cleave dsRNA [ 9 ]. Therefore fluo-
rescently labeled ssRNA can be used as control as well.
Bacterial RNase III generates Dicer-like termini and overhangs
after cleavage of dsRNA, but cleavage products differ in size. RNase
III of Escherichia coli cleaves dsRNA into 12-15-nucleotide-long
RNA fragments. Using GAPDH-derived dsRNA substrate
(Fig. 1a ) RNase III produces RNA fragments that differ in size
from Dicer products. Significant differences should be observed
comparing autocorrelation curves as well as diffusion coefficients
of cleavage products derived of both enzymes.

2 Materials

Prepare all solutions RNase free ( see Note 1 ).

1. Recombinant human Dicer (Genlantis).
2. E. coli RNase III (e.g., New England Biolabs).
3. Dicer substrates:
Sense RNA (5′-AAGGCUGAGAACGGGAAGCUUU-3′).
Antisense RNA 5′-ATTO647N (5′-ATTO647N-AGUUCC
GACUCUUGCCCUUCGAAAAG- 3′) ( see Note 2 ).
Antisense RNA 5′-Cy5 (5′-Cy5-AGUUCCGACUCUUGCC
CUUCGAAAAG-3′).
4. Buffer A: 30 mM Tris-HCl, pH 6.8, 50 mM NaCl.
5. Buffer B: 20 mM Tris-HCl, pH 8.0, 250 mM NaCl.
6. Buffer C: 10 mM Tris-HCl, pH 7.5, 350 mM KCl, 0.1 mM
EDTA, 0.1 mM DTT, 2.5 mM MgCl 2 .
7. 100 mM MgCl 2 stock solution.
8. 100 mM CaCl 2 stock solution.
9. RNase-free water.

2.1 Cleavage Assay

2.2 FCS
Measurement

1. Confocal laser scanning microscope (cLSM) with photon
counting unit (e.g., ConforCor2 (Carl Zeiss) with software
package) ( see Note 3 ).
2. Cover slip 24 × 50 mm (CarlRoth) ( see Note 4 ).