Double-Stranded Cyclic Oligonucleotides with Non-Nucleotide Bridges

Abstract

A series of double-stranded, cyclic oligodeoxynucleotides with non-nucleotide bridges have been synthesized, and their physicochemical properties and susceptibility to enzymes have been investigated. These bridged duplexes are of potential interest for their binding properties to transcription factors and other DNA-binding proteins. Triethylene glycol has been employed as the bridge to alter the lipophilicity of the duplex and avoid the potential for enzymatic cleavage. The synthetic route involved the synthesis of a 3'-phosphorylated, nicked double-stranded precursor with the final internucleotide bond being formed chemically using a water soluble carbodiimide. These bridged duplexes have high thermal dissociation temperatures, and the Tm for a triethylene-bridged 20 base pair duplex was higher than that for the corresponding pentathymidylate-bridged duplex. EcoR I endonuclease cleaved a ligated, bridged duplex at a slower rate than the corresponding unmodified duplex, whereas the unligated, bridged duplex was cleaved more rapidly. Sufficient amounts of the bridged octamer and dodecamer were prepared for proton NMR spectroscopic studies, and 2D COSY and NOESY spectra were obtained. The results indicate that the ligated duplex has a B-form conformation.

High-Purity Preparation of a Large DNA Dumbbell

“…The anomalously fast mobility of closed, double-ligated DNA dumbbells in the cold gel could be attributed to the wellknown fact of very high stability of their secondary structure with respect to melting (Erie et al, 1989;Ashley and Kushlan, 1991;Gao et al, 1994;Abe et al, 1998). We assume that because of this feature, the closed DNA dumbbell retains, at least partially, the duplex form under low-temperature denaturing conditions.…”

“…1), are of considerable interest for DNA biophysical/biochemical studies (Erie et al, 1989;Doktycz et al, 1992;Goldstein and Benight, 1992;Paner et al, 1993;Gao et al, 1994;Owczarzy et al, 1999). DNA dumbbells also have been considered to be promising oligonucleotide therapeutics acting as decoys for DNA-processing proteins (Chu and Orgel, 1991;Clusel et al, 1993;Hosoya et al, 1999).…”

“…The lower limit is imposed by steric and conformational constraints in small DNA dumbbells that compromise the efficiency of DNA ligase (Erie et al, 1987). Chemical syntheses were developed later for DNA dumbbell preparation (Ashley and Kushlan, 1991;Ippel et al, 1992;Dolinnaya et al, 1993;Gao et al, 1994;Kuznetsova et al, 1999), which overcame difficulties with making very small DNA dumbbells intrinsic to biochemical approaches, yielding DNA dumbbells with duplex stems as short as 3 bp.…”

Stabilization of double-stranded oligonucleotides using backbone-linked disulfide bridges

“…A second product from the oxidation of the disulfhydryl oligonucleotide 2 was also obtained in relatively high yield under certain conditions, and on the basis of its mobility on PAGE was determined to be the bridged hairpin structure 4. In order to confirm the structure of 3, this material was also prepared by an alternate route in which the two disulfide bridges were incorporated directly into the oligonucleotide. This procedure closely paralleled that previously employed for the synthesis of a triethylene glycol-bridged duplex (11) and involved the preparation of a self-complementary nicked intermediate 5 possessing two disulfide bridges and a 3' phosphomonoester group. The intermediate 5 was then chemically ligated using a carbodiimide coupling agent to produce the closed circular duplex 3.…”

“…The oligonucleotide 5 (6 OD260) was ligated using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride according to a previously described method (11) and isolated to give 3 (1.5 OD260). PAGE showed a single band with mobility identical to a sample prepared by the oxidative procedure as described above.…”