after mixing.Kis then plotted against the concentrations of
target oligonucleotides (Fig.1c).- To calculate effective affinityKD, linear regression is conducted
on allKvalues,K¼Kon[target oligo] +Koff.KDis calculated
asKD¼Koff/Kon(Fig.1c).
3.1.3 Validating
Sequence Selectivity
In Vitro
- To measure sequence-selective fluorescence activation, fluores-
cence spectra of the probes in the absence or presence of
complementary RNA/DNA is measured using a cuvette with
a 1-cm path length. - ECHO-liveFISH probes are mixed with equimolar (non)com-
plementary RNA/DNA molecules in Dilution buffer by vor-
texing, incubated up to 5 min in Eppendorf tubes and
transferred to cuvette for measurement (Fig.1e). - The mixture is excited at 514 nm (1.5 nm bandwidths) and
fluorescence emission between 535 and 700 nm is recorded. - A sum of fluorescence intensity between 535 and 700 nm is
calculated as the probe on–off ratio (on: with complementary
sequence; off: without complementary sequence) (Fig.1e).
3.2 Introducing ECHO
Probes into Living
Mouse Brain
Intact cell membranes are not permeable to ECHO probes, which
are water-soluble and hydrophilic; therefore, delivery methods
assisting ECHO probes to cross plasma membrane are required.
Thus, we have adapted an in vivo electroporation protocol that has
been used to deliver DNA plasmids into postnatal mouse cerebella
to study migration behavior of cerebellar cells [32]. This method
allows oligonucleotide probes to be directly injected into the brain
regions through a needle electrode (an anode, Fig.2a,seeNote 2).- Connect a pair of tweezer-type cathode electrodes with an
adaptor cable, hook-type anode electrode and a foot switch to
electrical pulse generator, and prepare sterilized surgery tools
(seeNote 3). - Stabilize an injection microsyringe with a 33-G needle
connected to the anode onto the micromanipulator at a tilting
angle of 60(Fig.2a). - Anesthetize a postnatal day 7–9 ICR mouse by covering its
entire body with ice for 6–7 min (seeNote 4). - Rinse the injection needle connected to the microsyringe by
drawing in and pushing out 70% EtOH and sterile water alter-
natively three times and load 0.8 μL of sample solutions
(ECHO probes or DNA plasmids) into the injection syringe
by pulling back the plunger. - Lay the anesthetized mouse on a surgery booster and mount
with a Band-Aid (Fig.2b).
264 Dan Ohtan Wang