Fluorescence Resonance Energy Transfer Analysis of the Degradation of an Oligonucleotide Protected by a Very Stable Hairpin

Abstract

In vitro degradation of antisense oligonucleotides protected or not on their 3' side against enzymatic attack by a naturally forming hairpin has been studied by fluorescence resonance energy transfer (FRET). The two oligonucleotides d(5"TTCTCGCGAAGC3') forming the hairpin and d(5"TTCTCCGGAAGC3') as a control were labeled on their 5' side by tetramethylrhodamine and on their 3' side by fluorescein. Fluorescein has been shown not to hinder the hairpin formation and to give an additional protection against nucleases. The FRET technique proved adequate for an in situ study of these protected antisense oligonucleotides in living cells.

Best Minimally Modified Antisense Oligonucleotides According to Cell Nuclease Activity

“…In the case of R in culture medium, the degradation t 1/2 is longer in FRET experiments, 30 minutes instead of 15 minutes. This could be due to the protection provided by fluorescein itself on the 39-end of R (Réfrégiers et al, 1996). In all cases, the t 1/2 of H degradation is longer than that of R degradation.…”

“…This difference is moderate: 1 hour for H in culture medium (2 hours for ODN-H in PAGE experiment) vs. 30 minutes for R (15 minutes for ODN-R in PAGE), 1 hour 45 minutes for H in NIH-MDR-G185 lysate vs. 1 hour 10 minutes for R (no difference for ODN-H and ODN-R in PAGE). The protection provided by the hairpin against 39-exonuclease attack has been well demonstrated (Hirao et al, 1994;Réfrégiers et al, 1996). Advance of 59-exonuclease along the oligonucleotide is likely hindered by the hairpin.…”

“…At the first enzymatic cleavage, FRET of a single oligonucleotide disappears. It is then possible to follow oligonucleotide degradation in solution by FRET disappearance, that is, increase in the I 520nm /I 577nm ratio (Réfrégiers et al, 1996). Figure 5 presents the degradation percentages of H and R as a function of time in culture medium or NIH-MDR-G185 or K562 lysates (0.6 mg/ml protein in both cases).…”

“…We have shown (Réfrégiers et al, 1996) that FRET (Förster, 1948) allows serum degradation of short oligonucleotides to be followed. Two dodecamer oligonucleotides that have (H) or have not (R) the self-forming hairpin at their 39-end (Table 1) on their 39-end.…”

“…Our starting point was an all-PS antisense oligonucleotide that has proven its efficiency against this gene (Alahari et al, 1996). The stability against serum 39-exonucleases of oligonucleotides protected at their 39-end by the sequence d(GCGAAGC) has been used (Hirao et al, 1994;Réfrégiers et al, 1996). This sequence spontaneously forms a hairpin known for its extraordinary stability with regard to thermal denaturation or nuclease degradation (Hirao et al, 1994;Khan and Coulson, 1993).…”