A cytosine analog that confers enhanced potency to antisense oligonucleotides

Abstract

Antisense technology is based on the ability to design potent, sequence-specific inhibitors. The G-clamp heterocycle modification, a cytosine analog that clamps on to guanine by forming an additional hydrogen bond, was rationally designed to enhance oligonucleotide/RNA hybrid affinity. A single, context-dependent substitution of a G-clamp heterocycle into a 15-mer phosphorothioate oligodeoxynucleotide (S-ON) targeting the cyclin-dependent kinase inhibitor, p27(kip1), enhanced antisense activity as compared with a previously optimized C5-propynyl-modified p27(kip1) S-ON and functionally replaced 11 C5-propynyl modifications. Dose-dependent, sequence-specific antisense inhibition was observed at nanomolar concentrations of the G-clamp S-ONs. A single nucleotide mismatch between the G-clamp S-ON and the p27(kip1) mRNA reduced the potency of the antisense ON by five-fold. A 2-base-mismatch S-ON eliminated antisense activity, confirming the sequence specificity of G-clamp-modified S-ONs. The G-clamp-substituted p27(kip1) S-ON activated RNase H-mediated cleavage and demonstrated increased in vitro binding affinity for its RNA target compared with conventional 15-mer S-ONs. Furthermore, incorporation of a single G-clamp modification into a previously optimized 20-mer phosphorothioate antisense S-ON targeting c-raf increased the potency of the S-ON 25-fold. The G-clamp heterocycle is a potent, mismatch-sensitive, automated synthesizer-compatible antisense S-ON modification that will have important applications in the elucidation of gene function, the validation of gene targets, and the development of more potent antisense-based pharmaceuticals.

Figure 1

(A) Chemical structure of heterocycle analogs. (B) Model of G-clamp hybridized to guanine within a RNA/oligonucleotide helix.

Figure 2

The bioactivity of the G-clamp-modified p27^kip1 S-ON is context-dependent. (A) Sequence and position of the G-clamp modification (G-clamp is denoted by X) tested for p27^kip1 antisense activity. All linkages are phosphorothioate. T, thymidine; G, 2′-deoxyguanosine; C, 2′-deoxy-5-methylcytidine. (B) Immunodetection of p27^kip1 and PI3K (p85 regulatory subunit) after transfection of CV-1 cells with GS3815 cytofectin alone (2.5 μg/ml; lane 1), or GS3815 cytofectin (2.5 μg/ml) complexed with 30 or 10 nM of ON1 (lanes 2 and 3), ON2 (lanes 4 and 5), ON3 (lanes 6 and 7), ON4 (lanes 9 and 10), ON5 (lanes 11 and 12), or ON6 (lanes 13 and 14). At 30 nM, ON1 inhibited p27^kip1 protein levels by 40%, ON2 by 72%, ON3 by 88%, ON4 by 98%, ON5 by 54%, and ON6 by 98%. No inhibition of p27^kip1 protein levels was observed for ON1, ON2, ON3, and ON5 at 10 nM. The two most potent S-ONs, ON4 and ON6, inhibited p27^kip1 protein levels by 84 and 57%, respectively, at 10 nM (lanes 10 and 14). PI3K was used as an internal control for protein integrity, concentration, and transfer variations among the samples.

Figure 3

The G-clamp-modified p27^kip1 S-ON demonstrates potent, dose-dependent antisense inhibition. Immunodetection of p27^kip1 and PI3K (p85 regulatory subunit) after transfection of CV-1 cells with GS3815 cytofectin (lane 1); C5-propynyl with 11 substitutions (ON8; lanes 2–4)(see Table 2 for sequences); 5MeC, 5-methylcytosine control (ON9; lanes 5–7); phenoxazine-modified (ON10; lanes 8–10), and G-clamp modified (ON4; lanes 11–13) using 2.5 μg/ml GS3815 cytofectin and a range of S-ON concentrations (30, 10, and 3 nM).

Figure 4

G-clamp-modified S-ONs demonstrate sequence-specific inhibition of p27^kip1. Immunoblot analysis of PI3K and p27^kip1 in CV-1 cells transfected with 2.5 μg/ml GS3815 cytofectin complexed in the absence (lane 1) or presence of a range of concentrations of the G-clamp-modified S-ON, ON4, (lanes 2–5), or 2-nt-mismatch G-clamp-modified S-ONs (lanes 6–9). The sequence for the 2-nt G-clamp p27^kip1 mismatch S-ON is 5′-TGG CTC XCT TGC GCC, where the X indicates the position of the G-clamp substitution and italics denotes the mismatch sites. The G-clamp-modified p27^kip1 antisense S-ON inhibited p27^kip1 by 98.8% at 90 nM, 95.4% at 30 nM, 81.0% at 10 nM, and 26.7% at 3 nM when normalized to PI3K and compared with p27^kip1 protein levels in the control lane (lane 1, GS3815 cytofectin only). No effects on p27^kip1 levels were seen in extracts treated with the 2-nt mismatch S-ON. At 90, 30, 10, and 3 nM of the mismatch S-ON, p27^kip1 was expressed at 112, 107, 119, and 100%, respectively, of the control lane.

Figure 5

Incorporation of a single G-clamp into an optimized c-raf antisense S-ON dramatically enhanced its antisense potency. T24 bladder carcinoma cells were transfected with GS3815 cytofectin (2.5 μg/ml) complexed with varying concentrations of an unmodified c-raf antisense (AS) S-ON; an unmodified 4-nt mismatch (MSM) S-ON; a G-clamp c-raf antisense (AS) S-ON; and a 4-nt G-clamp mismatch (MSM) S-ON. Note that the G-clamp modified c-raf S-ONs were tested at a 10-fold lower S-ON concentration than the unmodified c-raf S-ON. Forty-eight hours later, extracts were prepared and analyzed for c-raf levels by immunodetection. p34cdc2 was used as an internal control. At 250 nM (lane 2) and 100 nM (lane 4), the unmodified c-raf antisense S-ON demonstrated a reduction in c-raf protein levels of 62 and 29%, respectively, when normalized to p34cdc2 and compared with control c-raf levels (GS3815 cytofectin only, lane1). The unmodified 4-nt mismatch S-ON had no effect on c-raf protein levels (lanes 3 and 5). At 25 nM (lane 6) and 10 nM (lane 8), the G-clamp-modified c-raf antisense S-ON inhibited c-raf protein levels by 76 and 63%, respectively. The G-clamp-modified 4-nt mismatch S-ON showed a modest 20% decrease in c-raf protein levels at 25 nM of the S-ON (lane 7). No effect on c-raf protein levels was seen at 10 nM of the G-clamp modified 4-nt mismatch S-ON (lane 9).