Bioconjugate Chem. 1995, 6, 466-472
466
Preparation and Nuclease Activity of Hybrid “Metallotris(methy1pyridinium)porphyrin Oligonucleotide” Molecules Having a 3’-Loop for Protection against 3’-Exonucleases Beatrice Mestre, Genevieve Pratviel, and Bernard Meunier” Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, 31077 Toulouse Cedex, France. Received November 22, 1994@
A 5‘-GCGAAAGC minihairpin structure was added to the 3’-end of an oligonucleotide substituted at the 5’-end by a manganese cationic porphyrin in order to enhance the 3‘-exonuclease resistance of these cleaver-antisense molecules. The influence of this minihairpin on the 3’-exonucleaseresistance, the binding affinity to a target ssDNA, and the cleaving efficiency of Mn-cationic porphyrin oligonucleotide conjugates was compared to that of the parent molecule without the 3’-hairpin. The results showed that the 3’-hairpin slightly decreased the binding affinity and consequently the cleaving efficiency of the conjugated molecule toward a target sequence, but the much higher nuclease resistance makes 3‘-minihairpin-protectedmetalloporphyrin oligonucleotides good candidates as reactive antisense oligonucleotides for studies on cells.
INTRODUCTION
The inhibition of gene expression by modified oligonucleotides is currently a possible approach in the chemotherapy of cancers or viral diseases (1-61, One way to enhance the antisense activity of an oligonucleotide is to mediate irreversible damage on its target nucleic acid (RNA or DNA) sequence. Different transition metal complexes have been used as oxidative DNA and RNA cleavers and linked to oligonucleotide vectors: Fe-EDTA1(7, 81, Cu-OP(41, and metalloporphyrins (912). A hybrid “nucleic acid cleaver-oligonucleotide” suitable for possible development as a therapeutic agent must be able to cleave DNA or RNA with a high chemical yield (81, inside of selected cells (for recent articles on different methods to improve cell penetration by modified oligonucleotides, see 13-15). We focused our attention on cationic manganese porphyrin complexes as nucleic acid cleavers to be linked to oligonucleotides for the following reasons: (i) cationic metalloporphyrins have an affinity for dsDNA ranging from lo4 to lo6 M-’ (16, 17), (ii) the parent manganese(111) neso-tetrakis(4-N-methylpyridiniumyl)porphyrin, Mn-TMPyP, is a very efficient DNA cleaver able to hydroxylate carbon-hydrogen bonds of deoxyriboses accessible from the minor groove of B DNA (18, 19); (iii) these cationic metalloporphyrins provided good bleomycin models when attached to an intercalating agent (20,211, and these latter hybrid molecules also have an anti-HIV activity (22); and (iv) the tris(4-N-methylpyridiniumy1)porphyrinatomanganese(II1) motif (Mn-trisMPyP)is able Abstract published in Advance ACS Abstracts, July 1,1995. Abbreviations: CDI, N,”-carbonyldiimidazole; EDTA, ethylenediaminetetraacetic acid; HOAt, 1-hydroxy-7-azabenzotriazole; TEAA, triethylammonium acetate; MOPS: 3-(N-morpholin0)propanesulfonic acid; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; TRIS, tris(hydroxylmethy1)aminomethane; Mn-trisMPyP-oligonucleotide, tris(4-N-methylpyridiniumy1)porphyrinatomanganese(II1) conjugated to a n oligonucleotide; Mn-TMPyP, pentaacetate of meso-tetrakis(4-N-methylpyridiniumyUporphyrinatomanganese(II1);oP, ortho-phenanthroline; OD, optical density; FCS, fetal calf serum; ds: double-stranded; ss: single-stranded.
to cleave DNA and RNA of HIV-1 a t low concentrations (5-100 nM) (11,12,23)when tethered to the 5’-end of a 19-mer oligonucleotide complementary to the initiation region of tat. For in vitro experiments, the metalloporphyrin moiety is activated by potassium monopersulfate, KHSOs, a water-soluble peroxide able to generate metaloxo species in water solutions (24). We expect the i n vivo activation mode of these DNA cleavers to be similar to that of bleomycin; i.e., molecular oxygen and a reducing agent will be used inside of cells (25). So we intend to address the DNA/RNA cleaving activity of the cationic metalloporphyrin moiety (Mn-trisMPyP) for a selected RNA/DNA sequence i n vivo by the means of its covalent attachment to an antisense oligonucleotide. The Mnporphyrin cleaver is here positioned at the 5’-end of the oligonucleotide. The metabolic stability of oligonucleotides is low due to the action of nucleases (mainly 3’-exonucleases) in extracellular fluids and intracellular compartments. Chemical modifications of oligonucleotides have been described to improve their stability (1-61, but the sugarphosphate backbone chemical modification did not always improve the antisense activity, and little is known about the toxicity and the mutagenicity of the metabolites arising from oligomer analogues. One simple way to make an oligonucleotide resistant to 3’-exonucleases without any associated chemistry is to add a t the 3’-end of the antisense a defined short sequence in order to form a remarkable stable minihairpin structure (26-29) (see Chart 1). In the present work we compared the DNA cleavage efficiency of Mn-trisMPyP-oligonucleotide that carried a highly stable minihairpin at the 3’-end of the oligonucleotide vector (Chart 1, conjugate 2) with the “3‘unprotected” corresponding antisense (Chart 1, conjugate 1). We prepared the Mn-trisMPyP conjugates with some modifications of published procedures (12,301. The two antisense oligonucleotides shared the same 19-mer sequence. The 19-mer sequence is complementary to the initiation codon of the tat gene of HIV-1. The longer 27mer is 3‘-protected by a previously described stable minihairpin structure (26, 27) consisting of eight nucleotides added on its 3‘-end (5’-GCGAAAGC). The DNA cleavage activity was assayed on a ss-35-mer oligonucleo-
1043-1802/95/2906-0466$09.00/00 1995 American Chemical Society
Nuclease Activity of Mn-trisMPyP-Oligonucleotide Hybrids
Bioconjugate Chem., Vol. 6, No. 4, 1995 467
Chart 1. Structures of Mn-trisMPyP-19mer (1)and Mn-trisMPyP-27mer (2)”
R
3’
Conjugate 1, Mn-trisMPy P-19-mer oligonucleotide = GGCTCCATTTCTTGCTCTC-OH Conjugate 2, Mn-trisMPyP-27-mer oligonucleotide = GGCTCCATTTCTTGCTCTmA :: A ,
I
HO-CGA 3’
35-mer target = 3’-HO-ATCCTAGATGACCGAGGTAAAGAACGAGAGGAGAC-OH The manganese(II1) axial ligand is a water a t pH other side.
