Labeling of dsRNA with CypHer5E Fluorescent Dye.

The labeling of dsRNA with a pH-sensitive fluorescent cyanine dye (CypHer5E) was carried out as previously described.³⁴ Aminoallyl dsRNA was synthesized by in vitro transcription using MEGAscript T7 kit (Ambion, U.S.A.) according to the manufacturer’s instructions except that UTP was replaced with amino-allyl UTP (Thermo Fisher Scientific, U.S.A.). A 348 bp T7-GFP PCR product purified using Qiagen PCR purification columns followed by synthesized dsRNA with amino-allyl UTP. The purified dsRNA was conjugated with a pH-sensitive cyanine dye, CypHer5E mono-NHS ester (GE Healthcare, U.K.) with an excitation and emission wavelength of 644 and 663 nm, respectively. One vial of CypHer5E dye (1 mg) was dissolved in 40 mL of DMSO and divided into aliquots of 2 μL in Eppendorf tubes. The dye was dried using a freeze-dryer (Labconco Freezone 6) and stored at 40 °C until use. For conjugation of dye to dsRNA, one vial of dye (~50 mg) was resuspended in a mixture containing 2 μg of aminoallyl UTP-labeled dsRNA (in 3.33 μL H₂O), 5 μL of DMSO, and 1.66 μL of 0.3 M sodium bicarbonate buffer, pH 9.0. The conjugation reaction was carried in the dark for 1 h at room temperature. The conjugated dsRNA was repurified using Qiagen PCR purification columns and eluted in DEPC-treated water.