2'-O-methyl-modified phosphorothioate antisense oligonucleotides have reduced non-specific effects in vitro

Abstract

Antisense oligodeoxynucleotides (ODNs) have biological activity in treating various forms of cancer. The antisense effects of two types of 20mer ODNs, phosphorothioate-modified ODNs (S-ODNs) and S-ODNs with 12 2'-O-methyl groups (Me-S-ODNs), targeted to sites 109 and 277 of bcl-2 mRNA, were compared. Both types were at least as effective as G3139 (Genta, Inc.) in reducing the level of Bcl-2 protein in T24 cells following a 4 h transfection at a dose of 0.1 micro M. Circular dichroism spectra showed that both types formed A-form duplexes with the complementary RNA, and the melting temperatures were in the order of Me-S-ODN.RNA > normal DNA.RNA > S-ODN.RNA. In comparison with the S-ODN, the Me-S-ODN had reduced toxic growth inhibitory effects, was less prone to bind the DNA-binding domain A of human replication protein A, and was as resistant to serum nucleases. Neither type of oligomer induced apoptosis, according to a PARP-cleavage assay. Hybrids formed with Me-S-ODN sequences were less sensitive to RNase H degradation than those formed with S-ODN sequences. Despite this latter disadvantage, the addition of 2'-O-methyl groups to a phosphorothioate-modified ODN is advantageous because of increased stability of binding and reduced non-specific effects.

Figure 1

Reduction of Bcl-2 level in T24 cells by ODNs targeted to different sites on bcl-2 mRNA. The percent reductions were relative to the amount of Bcl-2 in mock-treated cells from western blots. Oligomer sequences and S-ODN and 5′-Me-S-ODN modifications are shown in Table 1. G3139 is an 18mer S-ODN positive control. Error bars are ±1 SD of at least three determinations. The ODN doses were 0.1 µM for Figures 1 and 3.

Figure 2

Measured CD spectra of ODN·RNA hybrids compared with the averaged spectra of the individual strands. Hybrids were formed by pairing the following ODNs with the complementary RNA-109 strand: (A) Unmodified ODN-109, (B) S-ODN-109, (C) 5′-Me-S-ODN-109. (Solid line) Measured spectra; (dashed line) averaged spectra of the individual, unpaired strands. Spectra were taken at 20°C with samples in 0.15 M K⁺ (phosphate buffer, pH 7.0).

Figure 3

Growth inhibition of T24 cells following treatments with the S-ODN and the 5′-Me-S-ODN targeted to site 109 on bcl-2 mRNA. Growth inhibition was determined as the decrease in [³H]thymidine incorporation, relative to the incorporation in mock-treated cells. Error bars are ±1 SD of three determinations.

Figure 4

Survival in FBS of oligomers 109. (A) Bands of intact ODNs in polyacrylamide gels after incubation in 10% FBS at 37°C for the indicated times. Bands were visualized by staining with SYBR Green II. (B) Percentages of uncleaved oligonucleotides as a function of incubation time.

Figure 5

Competition binding experiments. The relative binding affinity of RPA70-A for S-ODN and 5′-Me-S-ODN was determined by competition of (A) cold S-ODN-109 or (B) cold 5′-Me-S-ODN with the ³²P end-labeled S-ODN-109 in a preformed complex with RPA70-A. The percent dissociation of S-ODN from the complex was calculated from the band intensity of free ³²P-labeled S-ODN released from the ³²P S-ODN:RPA70-A complex. (C) ³²P end-labeled S-ODN-109 or ³²P end-labeled 5′-Me-S-ODN-109 were titrated with RPA70-A in the presence of complementary RNA. In lanes 1 and 5, there were ODNs only, without RNA. In the other lanes, RNA and RPA70-A were added and, after 15 min, the complementary ODNs were added. The [P]/[N] ratios are the RPA70-A protein molar concentrations per total molar concentrations of nucleotides (DNA plus RNA). Multiple bands of the ODN·RNA duplexes were probably the result of some degradation of the RNA combined with a reduced stability of hybrids formed with the S-ODN. The complexes of RPA70-A with 5′-Me-S-ODN were more diffuse than those with the S-ODN, likely due to multiple arrangements of up to five protein molecules on the chimeric oligomer.

Figure 6

RNase H elicitation of modified ODN·RNA duplexes. With the assumption of equal loading, the percent degradation was calculated from the reduction in the intensity of the DNA·RNA duplex band after RNase H treatment. The complementary 20mer RNAs were ³²P end-labeled. Bands labeled as degraded products in the RNase H negative lanes were from labeled fragments of RNA and over-represent the amount of degradation in the RNA samples because at least two fragments result from a single cleavage. They did not affect the calculation of percent degradation of hybrids by RNase H.