increase of the DNA intercalating dye thiazole orange (TO) [5].
TO is used as a base surrogate, replacing one of the internal bases of
a target specific oligonucleotide. Upon mismatch-free duplex for-
mation, the dye is positioned in a high viscosity microenvironment
that limits conformational changes of TO and thus activates its
fluorescence. Injecting as few as three different nuclease resistant
FIT probes we successfully visualized theoskartransport mRNAs in
developingDrosophilaoocytes [6]. A locked nucleic acid (LNA)
nucleotide adjacent to TO uniquely increased the brightness of the
FIT probes and also contributed to nuclease resistance. We
obtained identical results of the RNP transport properties to that
measured with the reference oskar-MS2(10x), MCP-EGFP (or
MCP-mCherry) transgenic system. The different fluorescence
spectrum of TO, EGFP (or mCherry) fluorescent proteins allowed
the simultaneous visualization of two labels. Moreover, targeting
the localization element (LE) of the endogenous transcript that is
responsible for the intra-ooplasmic, kinesin-1 mediated transport
ofoskarmRNPs [7] with a FIT-probe, we could recapitulate the
findings of our previous transgenic mutagenesis analysis while hav-
ing visual confirmation on the probe–target duplex formation and
the consequent disruption of the LE [6]. Here, we describe the
probe design, synthesis and analysis procedures as well as the
microinjection and live cell imaging of developing Drosophila
oocytes.2 Materials
Prepare all solutions using ultrapure water (ddH 2 O) and analytical
grade reagents unless indicated otherwise. Follow waste disposal
and safety regulations as indicated on the reagent storing
containers.2.1 Probe Synthesis 1. 3^0 -Spacer-C3-CPG (1μmol, pore size 500 A ̊) and 2’O-Me-
RNA phosphoramidites (e.g., Link Technologies).
- DNA phosphoramidites (e.g., Thermo Fisher Scientific).
- LNA phosphoramidites (e.g., Exiqon).
- Dye monomer synthesis is carried out according to F. Ho ̈vel-
mann et al.,Chem.Sci., 2016 , 7 , 128–135. - DNA synthesizer (e.g., MerMade-4 Synthesizer Bioautomation).
- Activator: Hyacinth DMT-solution (e.g., emp Biotech).
- Capping A: THF–lutidine–acetic anhydride (e.g., emp
Biotech). - Capping B: 10 v/v % 1-methylimidazole in THF (e.g., emp
Biotech).
274 Jasmine Chamiolo et al.