Oligonucleotide⁻Palladacycle Conjugates as Splice-Correcting Agents

Abstract

2'-O-Methylribo phosphorothioate oligonucleotides incorporating cyclopalladated benzylamine conjugate groups at their 5'-termini have been prepared and their ability to hybridize with a designated target sequence was assessed by conventional UV melting experiments. The oligonucleotides were further examined in splice-switching experiments in human cervical cancer (HeLa Luc/705), human liver (HuH7_705), and human osteosarcoma (U-2 OS_705) reporter cell lines. Melting temperatures of duplexes formed by the modified oligonucleotides were approximately 5 °C lower than melting temperatures of the respective unmodified duplexes. The cyclopalladated oligonucleotides functioned as splice-correcting agents in the HeLa Luc/705 cell line somewhat more efficiently than their unmodified counterparts. Furthermore, the introduction of this chemical modification did not induce toxicity in cells. These results demonstrate the feasibility of using covalently metalated oligonucleotides as therapeutic agents.

Keywords: oligonucleotide; organometallic; palladacycle; palladium; splice-correction.

Scheme 1

Cyclopalladation of oligonucleotides ON1b and ON2b. Reagents and conditions: (a) Li₂PdCl₄, MeCN, H₂O, 25 °C, 24 h.

Figure 1

UV melting profiles for duplexes formed by the RNA ON3 (A) and DNA ON4 (B) targets with ON1a (cyan circles), ON1b (magenta triangles), and ON1b-Pd (yellow squares); pH = 7.4 (20 mM cacodylate buffer); [oligonucleotides] = 1.0 μM; I(NaClO₄) = 0.10 M.

Figure 2

Restoration of Luciferase activity in HeLa Luc/705 cells by ON1a (cyan), ON1b (magenta), ON1b-Pd (yellow), ON2b (green), and ON2b-Pd (grey). The oligonucleotides were delivered by either lipofection (A) or gymnosis (B). Each column represents the mean with the standard error of the mean (SEM) of three (lipofection) or two (gymnosis) independent experiments (n ≥ 3). P-values were calculated by two-way ANOVA test and differences were statistically compared using post hoc Fisher’s LSD test (n.s. non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001).

Figure 3

Restoration of Luciferase activity in HuH7_705 (A,C) and U-2 OS_705 (B,D) cells by ON1a (cyan), ON1b (magenta), ON1b-Pd (yellow), ON2b (green), and ON2b-Pd (grey). The oligonucleotides were delivered by either lipofection (A,B) or gymnosis (C,D). Each column represents the mean with the standard error of the mean (SEM) of at least three independent experiments (n ≥ 3).

Figure 4

Viability of (A) HeLa Luc/705 and (B) HuH7_705 cells upon lipofection by oligonucleotides ON1a (cyan), ON1b (magenta), ON1b-Pd (yellow), and ON2b-Pd (grey). Each column represents the mean with the standard error of the mean (SEM) of two independent experiments (n = 2).