Abstract:The chemical ligation of 17 50-54-membered nicked DNA dumbbells with different closing fragments, nick positions, and nucleotides facing the nick were investigated. T4, T5, GTA4C, GCGA2GC, and GCGA3GC sequences were chosen as the closing fragments. The nicks were placed in the center of the duplex stem or were adjacent to the closing fragments. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and cyanogen bromide were used as the condensing agents. We showed that the ligation efficiency is 10%-90% depending on t… Show more
“…As assessed from the intensities of each band in lane 6, annealing and ligation of ODN1 and ODN2 gave 72% of closed DNA dumbbell, whereas 25% of monoligated by-products and 3% of unligated reactants were observed. In accordance with prior similar observations with shorter DNA dumbbells (Kuznetsova et al, 1999), the closed large DNA dumbbell we assembled migrates much more slowly in the hot gel than does the corresponding nicked DNA dumbbell, in contrast to reversed relative mobilities of these species in the cold gel.…”
Section: Preparation and Initial Characterization Of Large Dna Dumbbesupporting
confidence: 92%
“…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.…”
We report on the efficient biochemical synthesis of a large DNA dumbbell starting from a pair of short DNA hairpins with long single-stranded tails of arbitrary sequence. The DNA dumbbell is obtained by enzymatic ligation yielding a 94-bp duplex stem closed at both termini by single-stranded loops of 5 nt. Following ligation, all unligated precursors and monoligated by-products were multiply biotinylated via nick-translation or primer-extension or both. Thus, they could readily be removed from the DNA dumbbell preparation by a mild biomagnetic separation procedure. The closed conformation of the purified DNA dumbbell was verified by its altered gel mobility as compared with unligated or monoligated samples and by an exonuclease assay. Considering the promising therapeutic potential of DNA dumbbells, the developed biosynthetic approach could be used for high-purity preparation of longer, covalently closed DNA decoys.
“…As assessed from the intensities of each band in lane 6, annealing and ligation of ODN1 and ODN2 gave 72% of closed DNA dumbbell, whereas 25% of monoligated by-products and 3% of unligated reactants were observed. In accordance with prior similar observations with shorter DNA dumbbells (Kuznetsova et al, 1999), the closed large DNA dumbbell we assembled migrates much more slowly in the hot gel than does the corresponding nicked DNA dumbbell, in contrast to reversed relative mobilities of these species in the cold gel.…”
Section: Preparation and Initial Characterization Of Large Dna Dumbbesupporting
confidence: 92%
“…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.…”
We report on the efficient biochemical synthesis of a large DNA dumbbell starting from a pair of short DNA hairpins with long single-stranded tails of arbitrary sequence. The DNA dumbbell is obtained by enzymatic ligation yielding a 94-bp duplex stem closed at both termini by single-stranded loops of 5 nt. Following ligation, all unligated precursors and monoligated by-products were multiply biotinylated via nick-translation or primer-extension or both. Thus, they could readily be removed from the DNA dumbbell preparation by a mild biomagnetic separation procedure. The closed conformation of the purified DNA dumbbell was verified by its altered gel mobility as compared with unligated or monoligated samples and by an exonuclease assay. Considering the promising therapeutic potential of DNA dumbbells, the developed biosynthetic approach could be used for high-purity preparation of longer, covalently closed DNA decoys.
“…In this case the chemistry used 3‘- or 5‘-phosphorylated DNAs combined with BrCN/imidazole/Ni 2+ as a condensing reagent (Scheme 4). , The circular DNAs were designed to bind to complementary RNAs and DNAs, and templates contained the same sequence that the circles were eventually intended to bind. Later studies by Turnbull further optimized these cyclizations and also addressed the converse situation, in which circular DNAs templated the BrCN-mediated ligation of linear oligonucleotides. , Circular “dumbbell” oligonucleotides, which are DNA duplexes with closed ends, have been chemically closed in self-templated reactions pursued by several laboratories. ,, …”
“…15, 16 Cyanogen bromide has been widely used for the chemical ligation of nicked duplexes and dumbbells. 17 Chemical ligation of nicked dumbbells or duplexes requires that the reactive groups be in reasonably close proximity, as is the case for the 5 -hydroxyl and 3 -phosphate groups in the minimized structure for 6N shown in Fig. 6.…”
Section: Synthesis and Structure Of The Dumbbellsmentioning
The synthesis and properties of nicked dumbbell and dumbbell DNA conjugates having A-tract base pair domains connected by rod-like stilbenedicarboxamide linkers are reported. The nicked dumbbells have one to eight dA-dT base pairs and are missing a sugar-phosphate bond either between the linker and a thymine nucleoside residue or between two thymine residues. Chemical ligation of all of the nicked dumbbells with cyanogen bromide affords the dumbbell conjugates in good yield, providing the smallest mini-dumbbells prepared to date. The dumbbells have exceptionally high thermal stability, whereas the nicked dumbbells are only marginally more stable than the hairpin structures on either side of the nick. The structures of the nicked dumbbells and dumbbells have been investigated using a combination of circular dichroism spectroscopy and molecular modeling. The base pair domains are found to adopt normal B'-DNA geometry and thus provide a helical ruler for studies of the distance and angular dependence of electronic interactions between the chromophore linkers.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
