Oxetane modified, conformationally constrained, antisense oligodeoxyribonucleotides function efficiently as gene silencing molecules

Abstract

Incorporation of nucleosides with novel base-constraining oxetane (OXE) modifications [oxetane, 1-(1',3'-O-anhydro-beta-d-psicofuranosyl nucleosides)] into antisense (AS) oligodeoxyribonucleotides (ODNs) should greatly improve the gene silencing efficiency of these molecules. This is because OXE modified bases provide nuclease protection to the natural backbone ODNs, can impart T(m) values similar to those predicted for RNA-RNA hybrids, and not only permit but also accelerate RNase H mediated catalytic activity. We tested this assumption in living cells by directly comparing the ability of OXE and phosphorothioate (PS) ODNs to target c-myb gene expression. The ODNs were targeted to two different sites within the c-myb mRNA. One site was chosen arbitrarily. The other was a 'rational' choice based on predicted hybridization accessibility after physical mapping with self-quenching reporter molecules (SQRM). The Myb mRNA and protein levels were equally diminished by OXE and PS ODNs, but the latter were delivered to cells with approximately six times greater efficiency, suggesting that OXE modified ODNs were more potent on a molar basis. The rationally targeted molecules demonstrated greater silencing efficiency than those directed to an arbitrarily chosen mRNA sequence. We conclude that rationally targeted, OXE modified ODNs, can function efficiently as gene silencing agents, and hypothesize that they will prove useful for therapeutic purposes.

Figure 1

Chemical structure of OXE modification and the DPPZ moiety.

Figure 2

Delivery of fluorescein labeled unmodified ODNs into K562 cells with the nucleoporation technique: phase (A) and fluorescent (B) low power (200×) photomicrographs.

Figure 3

QTR-PCR assay performed on RNA isolated from K562 cells transfected with OXE and PS antisense (AS) ODN molecules. Data are presented as a function of c-myb mRNA copies relative to 18S RNA. As noted above, 1 × 10⁶ K562 cells were suspended in 100 μl of nucleofection buffer solution with 5 μg of ODNs (final concentrations of ODNs during the nucleofection procedure were 5.2 μM for the 24mers, and 4.3 μM for 30mers). Control cells were subjected to nucleoporation, but in the absence of ODNs. OXE1 and PS 1 have identical sequence (24mer). OXE2, OXE3 and PS 2 are also sequence identical. Based on the mapping procedure described, these ODNs were predicted to hybridize with their target with high efficiency. OXE 2 and OXE 3 differ in number of oxetane modified nucleosides per molecule (four and five, respectively). Scrambled sequences (SCR 1 and 2) have the same nucleotide composition as OXE1 and OXE 2 respectively but in a random order.

Figure 4

Western blot on lysates from K562 control and transfected cells. About 1 × 10⁶ K562 cells were suspended in 100 μl of nucleofection buffer solution with 5 μg of ODNs (final concentrations of ODNs during the nucleofection procedure were 5.2 μM for the 24mers, and 4.3 μM for 30mers). Cells were lysed and 100 μg of total protein extract was resolved on polyacrylamide gel. The proteins were than transferred to PVDF membrane and probed first with antibody against c-myb (A), then the same membrane was stripped and probed again with antibody against beta actin (B). The intensity of bands was measured with a densitometer and the ratio of c-myb versus beta actin is presented in the graph (C).

Figure 5

Cell proliferation after electroporation of OXE and PS modified ODNs. About 3 × 10⁵ K562 cells were suspended in 100 μl of nucleofection buffer solution with 5 μg of the respective ODNs. Following electroporation cells were washed and then transferred to 12 well costar plates (Corning Inc., Corning, NY), each well containing 1.5 ml of RPMI with 10% FBS. An aliquot of cells were removed every 24 h over 4 days and counted with a hemocytometer.

Figure 6

Quantitation of ODNs delivered to cells by nucleoporation. K562 cells were nucleoporated in buffer alone (Control), or with PS or OXE ODNs. Five minutes later, the cells were lysed and the supernatant was separated from the residual cellular material by centrifugation. The resulting supernatant and cell pellet was then transferred to the membrane, and the blots were probed with a radiolabeled complementary sequence. (A) Slot blot of ODNs present in K562 cell supernatants, and residual cell pellet. Rows 1 and 2 of the upper blot are, respectively, PS and OXE ODN standards applied to the membrane for quantitation purposes. Columns labeled on the lower half of the blot represent material found in the cell supernatant and pellet, obtained from untreated control cells and cells nucleoporated with PS and OXE ODNs. (B) Densitometry analysis of slot blot shown in (A) above. Open columns represent material present in the cell supernatant and black columns represent material present in the cell pellet of PS and OXE treated cells.

Figure 7

Effect of DPPZ moiety on intracellular delivery of ODNs. K562 cells were nucleoporated in buffer alone (control), or with PS, OXE, or natural backbone ODNs with a 3′-DPPZ moiety. Five minutes later, the cells were lysed and the resulting material was then slot blotted to a membrane, which was then probed with a radiolabeled complementary sequence. The densitometry measurement of DNA (ng) found on the slot blot corresponding to control, OXE, natural backbone 3′-DPPZ and PS treated cells are shown.