A Fluorescence Correlation Spectroscopy-Based Enzyme Assay for Human Dicer - miRNA Maturation: Methods and Protocols - page 101

3 Methods

3.1 RNA
Denaturation
and Renaturation

1. Dilute ATTO647N-labeled RNA in Buffer A and combine it
with sense RNA equimolecular.
2. Dilute Cy5-labeled RNA in Buffer B and combine it with sense
RNA equimolecular.
3. Heat RNA for 3 min at 95 °C.
4. Cool sample down by 1 °C steps per 1.2 min until a tempera-
ture of 25 °C is reached.
5. Add MgCl 2 to a final concentration of 2.5 mM ( see Note 5 ).

3.2 FCS
Configuration for
ATTO647N and Cy5
(See Note 6)

1. Excite with 3 % of 5.0 mW of a HeNe laser at 633 nm.
2. Use the water immersion objective C-Apochromat 40× with
numerical aperture of 1.2.
3. Separate emitted light from detection volume with a 633 nm
main dichromic mirror (HFT) and 650 nm emission long-pass
filter.
4. Adjust pinhole to 90 μm.
5. Measurement period: 5 or 10 s.

3.3 FCS
Measurements

Measurements were carried out in a sample volume of 20 μl posi-
tioned on cover slide in cLSM at room temperature.

1. Measure diffusion time of 5 nM fluorescent dsRNA in the
absence of enzyme in appropriate buffer.
2. Add Dicer to a final concentration of 1 U directly to the sub-
strate on cover slide, and measure diffusion time again.
Diffusion time decreases significantly due to generation of
cleavage product ( see Note 7 ).
3. Plot autocorrelation curves of substrate and Dicer product
against diffusing time and compare (Fig. 2 ). The smaller the
molecular weight of a diffusing component the more the auto-
correlation curve is shifted to the left.
4. Optional control experiment 1: Perform Dicer assay with
another Dicer substrate, which is labeled with an alternative
fluorophore.
5. Optional control experiment 2: After RNA denaturation and
renaturation add CaCl 2 to a final concentration of 2.5 mM
instead of MgCl 2 to RNA ( see Note 8 ). Then perform Dicer
assay as described in steps 1 - 3 . No reduction of diffusion time
should be observed.
6. Optional control experiment 3: Perform Dicer assay with
single-stranded fluorescent RNA as substrate. No reduction
regarding diffusion time should be observed ( see Note 9 ).