Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET)

Abstract

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 microM and was not appreciably affected by the presence of non-specific DNA. The pre-formed hybrid slowly dissociated (T1/2 approximately 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.

Hybridization kinetics is different inside cells

“…S5B). Both strands were evenly distributed over the cytoplasm and showed an enhanced concentration inside the nucleus (26). An overlay of donor and acceptor images showed that the two strands colocalized.…”

“…A BLAST search was performed with the sequence of the 16-mer to minimize specific interactions with genomic DNA or messenger RNA. Each strand was capped at both ends by an enantiomeric cytosine (L-nucleotide) (25) to suppress degradation by exonucleases (26). Lipofection was used for DNA delivery into the cells (26).…”

Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection

“…At different times after incubation of lipidic vectors with mouse serum at 37°C, an aliquot of sample was added to PBS to a final volume of 3 ml and the emission spectra were recorded between 500 and 650 nm with excitation at 470 nm and 10 nm excitation and 6 nm emission slits. 29 Sensitivity of DNA to mouse serum Lipopolyplexes or lipoplexes of different lipid compositions were prepared as described. They were then mixed with mouse serum at a ratio of 1:2 (v/v) and the mixtures were incubated at 37°C with gentle shaking.…”

Characterization of the Nuclear Export Signal of Human T-Cell Lymphotropic Virus Type 1 Rex Reveals that Nuclear Export Is Mediated by Position-Variable Hydrophobic Interactions

“…Two days prior to injection, a confluent flask of Cos7 cells grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum was trypsinized, diluted 1:6 or 1:12, and seeded onto 25-mmdiameter glass coverslips. Cells were mounted into a custom temperature-controlled chamber and placed atop an inverted microscope equipped with microinjection and video monitoring equipment and a confocal scanning laser module (28). Approximately 50 fl of BSA-peptide conjugate was injected into the nuclei of Cos7 cells.…”