INTRACELLULAR OLIGONUCLEOTIDE HYBRIDIZATION DETECTED BY FLUORESCENCE RESONANCE ENERGY-TRANSFER (FRET)

Fluorescence resonance energy transfer (FRET) was used to study hybrid formation and dissociation after microinjection of oligonucleotides (ODNs) into living cells. A 28-mer phosphodiester ODN (+ PD) was synthesized and labeled with a 3' rhodamine (+ PD-R). The complementary, antisense 5'-fluorescein labeled phosphorothioate ODN (- PT-F) was specifically quenched by addition of the + PD-R. In solution, the - PT-F/+ PD-R hybrid had a denaturation temperature of 65 +/- 3 degrees C detected by both absorbance and FRET. Hybridization between the ODNs occurred within 1 minute at 17 mu M and was not appreciably affected by the presence of non-specific DNA. The pre-formed hybrid slowly dissociated (T-1/2 approximate to 3 h) in the presence of a 300-fold excess of the unlabeled complementary ODN and could be degraded by DNAse I. Upon microinjection into the cytoplasm of cells, pre-formed fluorescent hybrids dissociated with a half-time of 15 minutes, which is attributed to the degradation of the phosphodiester. Formation of the hybrid from sequentially injected ODNs was detected by FRET transiently in the cytoplasm and later in the cell nucleus, where nearly all injected ODNs accumulate. This suggests that antisense ODNs can hybridize to an intracellular target, of exogenous origin in these studies, in both the cytoplasm and the nucleus.