Structural basis for the RNA binding selectivity of oligonucleotide analogues containing alkylsulfide internucleoside linkages and 2′-substituted 3′-deoxyribonucleosides

Abstract

INTRODUCTIONIn this report we describe the synthesis of oligonucleotides containing sulfide-linked dinucleoside units, namely rT(2'OH)dT, rT(2'0Me)5dT, dT8rU(2'OMe) and dT(2o0Me)5rU(2oMe). We also describe the interactions of such oligomers with complementary DNA and RNA targets, and provide the structural basis for their remarkable RNA binding selectivity. In all cases, the Tm values of the SIP-chimera duplexes were lower than those of the corresponding unmodified duplexes. We attribute this to steric interactions between the 5' sulfur and the atoms of the nearby base/sugar residues. The 2'-substituents (i.e., 2'OH or 2'OMe) vicinal to the alkylsulfide internucleoside linkage significantly perturb the structure and stability of the duplexes formed with DNA, and more so than with RNA. The introduction of three rT(2'OH)sdTp ( ' (5,6).Our research in the antisense field has focused on oligonucleotide analogues in which some of the phosphodiester groups are replaced by the dialkyl sulfide linkage 3'-CH2-CH2-S-CH2-5' (7-11). This linkage is non-hydrolyzable, non-ionic, and its length approximates that of a phosphodiester linkage reasonably well. More recently, we focused attention on the preparation of rT(20OH)SdT, rT(2 OMe)SdT, dTsrU(2'OMe) and rT(2'OMe)SrU(2'OMe) dimer units as well as their 3'-O-phosphoramidite derivatives for incorporation into oligonucleotides (11,12 (55°C, 16 h). The ammonia solution was collected by centrifuging the Eppendorf tube and then run through a Sephadex NAP-10 column which was pre-washed with 15 ml deionized water. The first eluting fractions (1.5 ml) were lyophilized to dryness and the resulting oligomer was redissolved in 1 ml of water. Purity of each oligomer was confmed by PAGE (26% polyacrylamide-7 M urea) run at 4°C, at a current of 10 mA, for 6-8 h (9). Melting experimentsMelting curves were measured in 10 mM NaH2PO4/Na2HPO4, 1 M NaCl buffer (pH 7.0) and analyzed the same way as described in reference 9. Circular dichroismThe CD spectrum ofeach sample was measured on a Jasco 500-C spectropolarimeter with a Jasco DP-J800/300 data processor. The cell temperature was kept at 50C. The concentration of the oligonucleotides were -2.5 jM (each strand).

Synthesis and Thermal Denaturation Studies of Conformationally Restricted 3′‐C‐Ethynyl‐3′‐O,4′‐C‐methyleneribonucleotides

“…Similar conventions apply for the corresponding hydrogen atoms. Quaternary carbons were not assigned in the 13 C NMR spectra. Traces of solvents in NMR spectra were identified by reference to published data.…”

“…After column chromatography, appropriate fractions were pooled, evaporated and dried under high vacuum for at least 12 h to give the products in high purity (Ͼ95 %). 13 C NMR or 31 P NMR spectroscopy ascertained the sample purity.…”

“…1 H NMR, 13 C NMR and 31 P NMR spectra were recorded at 300 MHz, 75.5 MHz and 121.5 MHz, respectively. Chemical shifts are reported in parts per million (ppm) relative to deuterated solvent as the internal standard (δ H : [D 6 ]DMSO 2.50 ppm; δ C : [D 6 ]DMSO 39.43 ppm).…”

“…The preferred furanose ring conformation for 2Ј,5Ј-linked RNA is C2Ј-endo (South-type), [10][11][12] whereas it is C3Ј-endo (North-type) for 2Ј,5Ј-linked DNA strands against normal DNA or RNA. [11,13] In contrast, the furanose rings of normal 3Ј,5Ј-linked RNA or DNA strands preferentially adopt C3Ј-endo and C2Ј-endo conformations, respectively. [14] Modification of normal DNA strands with conformationally restricted nucleosides such as LNAs [15][16][17] in which the furanose ring is locked in a C3Ј-endo conformation has proven to be a very powerful strategy for achieving strong hybridization with normal DNA or RNA complements.…”

Chemical Stability of Nucleic Acid–Derived Drugs

“…[119][120][121][122][123] Despite its advantages regarding enzymatic stability, the thioether linkage 3Ј-CH 2 -CH 2 -S-CH 2 -5Ј is a potential candidate for oxidative attack on the thioether sulfur with all its consequences, such as formation of sulfur-centered radical cations, deprotonation, and fragmentation. These reactions are well known from various model studies regarding the oxidation mechanisms of organic sulfides.…”