(antisense/antigene/hybrid duplex/triple helix)

Deoxyribonucleic guanidine is a potential antisense agent that is generated via the replacement ofthe negative phosphodiester linkages ofDNA [-O-(POj)-0-O with positively-charged guanidinium (g) linkages [-NH--C(=NH2) -NH-]. A pentameric thymidyl deoxyribonucleic guanidine molecule [d(Tg)4T-azido] has been shown to base pair specifically to poly(rA) with an unprecedented affinity. Both double and triple strands consisting of one and two equivalents of d(Tg)4T-azido paired with one equivalent of poly(rA) are indicated by thermal denaturation experiments. At an ionic strength of 0.22, the five bases of d(Tg)4T-azido are estimated to dissociate from a double helix with poly(rA) at > 100°C! The effect of ionic strength on thermal denaturation is very pronounced, with stability greatest at low ionic strengths. The method of continuous variation indicates that there is an equilibrium complex with a molar ratio of d(Tg) to r(Ap) or d(Ap) of 2:1. Based on this evidence, models of the structures of d(Tg)9T-azido bound to r(Ap)9A are proposed. Putative drugs consisting of oligonucleotide analogs capable of arresting cellular processes at the translational or transcriptional level via base pair interactions with RNA or DNA are known as antisense and antigene agents, respectively (1-5). The backbones of viable antisense/antigene agents use linkages other than phosphodiesters due to the susceptibility of the backbones of RNA and DNA to degradation by cellular nucleases. To be effective, such agents must bind with fidelity to target nucleic acid sequences via Watson-Crick and Hoogsteen base pairing. Since antisense/antigene agents must compete with specific oligonucleotides and proteins for RNA/ DNA targets, it is desirable that these agents have a high affinity for their complementary sequences. The stability of doubleand triple-stranded RNA and DNA would increase if the electrostatic repulsion among the polyanionic single strands could be alleviated. This is seen in the enhanced binding of the noncharged peptide nucleic acids to singlestranded DNA (6, 7). One might suspect, therefore, that a strand of bases complementary to a nucleic acid sequence but connected together by positively charged linkages would act as a particularly effective antisense/antigene agent, since the repulsive effects found in duplexes of complementary nucleic acid strands would be replaced by attractive electrostatic interactions. Conversely, the electrostatic bonding between polycationic and polyanionic structures might be quite nonspecific and independent of complementary base pairing. Recently we described the synthesis and binding properties of the pentameric thymidyl deoxyribonucleic guanidine (DNG) d(Tg)4T-azido (1) (8-10), where g indicates a guanito linkage instead of a phosphate (p). Guanidinium groups [-NH-C(=NH )-NH-] replace the phosphodiester linkages [(-O--PO-)-O-] ofDNA to yield DNG. Both double and triple helical structures were formed between 1 and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1 poly(dA). No helical structures were observed between 1 and poly(dG), poly(dC), poly(dT), or poly(dI). Most noteworthy was the unprecedented stability of the DNG-DNA complex. A double helix containing one equivalent of pentameric thymidyl DNG and one equivalent of poly(dA) does not dissociate in boiling water at near-physiological ionic strength. Our present studies focus on the potential of DNG as an antisense agent. The results of thermal denaturation and hybridization studies between DNG and RNA are provided as are modeling schemes representing anticipated structures of DNG-RNA complexes. MATERIALS AND METHODS Synthesis. Synthesis of d(Tg)4T-azido (1) was as described (10). Thermal Denaturation Studies. Plots ofA260 vs. t (°C) for 1 in the presence of poly(rA), poly(rG), poly(rI), poly(rC), or poly(rU) were obtained at pH 7.0 (0.01 M KHP04 buffer) using a Perkin-Elmer UV/visible spectrophotometer in conjunction with a National Institute of Standards and Technology digital thermometer (accurate to ±0.2°C). The concentration of each of the oligonucleotides was 41.7 ,tM in bases. Concentrations of 1 and the polyoligonucleotides were determined spectrophotometrically from molar (nucleotidyl unit) extinction coefficients [8700 M-1 cm-1 at 268 nm for 1 (11), 9800 M-1 cm-1 at 258 nm for poly(rA), 10,400 M-1-cm-1 at 253 nm for poly(rG), 10,200 M-1 cm-1 at 248 nm for poly(rI), 6200 M-1-cm-1 at 269 nm for poly(rC), and 9350 M-1-cm-1 at 260 nm for poly(rU); values for the polyribonucleotides are Abbreviations: DNG, deoxyribonucleic guanidine; tm, thermal denaturation temperature; ABNR, adopted basis Newton-Raphson.