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APPLICATION NOTE
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dgRNA ID
(crRNA, 42 nt;
varying tracrRNA
length, < 90 nt)
sgRNA ID
(> 90 nt) K
d
(pM)
dgRNA2 — 5170 ± 906
dgRNA3 — 5181 ± 975
dgRNA4 — 12.9 ± 3.9
dgRNA5 — 7.2 ± 3.0
dgRNA6 — 6.1 ± 2.4
dgRNA7 — 6.6 ± 5.5
dgRNA8 — 6.6 ± 3.3
dgRNA9 — 1.3 ± 2.0
dgRNA10 — 8.8 ± 3.9
dgRNA11 — 5.5 ± 3.0
dgRNA12 — 12.6 ± 4.5
dgRNA13 — 6.1 ± 2.7
sgRNA7 7.3 ± 3.5
Table 2: Affinities of various crRNA/tracrRNA hybrids and a sgRNA as determined in
MST measurements using the Monolith™ NT.115Pico instrument. MST experiments
were performed with 100 pM Cy5-RNA at 50 % LED and 60 % MST power.
Material and Methods
RNA Synthesis, Cy5 labeling and hybridization
All RNAs were provided by Axolabs GmbH (Kulmbach,
Germany) and were made by automated chemical
solid-phase synthesis using commercially available
phosphoramidites. A er cleavage from the solid
support and deprotection, the synthesized RNAs were
purified by high performance liquid chromatography
(HPLC). For labeling of guide RNAs, a Cy5-conjugated
probe (Axolabs GmbH; Kulmbach, Germany)
complementary to the target binding site of the
crRNAs (in dgRNAs) or the sgRNAs was used. This
hybridization was performed using a 10 % molar
deficiency of the Cy5-labeled probe. The Cy5-labeled
crRNAs were finally hybridized to the tracrRNAs in
an equimolar ratio. In general, hybridizations were
carried out in 1 x phosphate buffered saline (PBS;
pH 7.4) by heating samples to 70 °C followed by slow
cooling down to room temperature.
Cas9 labeling
The maleimide-NT647 dye (Cat.# MO-L004) was used
for the labeling of Cas9. For buffer exchange and
removal of the residual dye, the Antibody Labeling Kit
columns were used (Cat.# MO-L007) according to the
manufacturer's instructions. For labeling, 3 μM of Cas9
NLS from S. pyogenes (M0641, New England Biolabs)
were mixed with 15 μM of maleimide-NT647 dye and
incubated in the dark at room temperature for 30 min.
A er removal of residual dye, labeled protein was
centrifuged at 10 000 g for 10 min at 4 °C.
