Solution structure and dynamics of DNA duplexes containing the universal base analogues 5-nitroindole and 5-nitroindole 3-carboxamide

Abstract

Universal bases hybridize with all other natural DNA or RNA bases, and have applications in PCR and sequencing. We have analysed by nuclear magnetic resonance spectroscopy the structure and dynamics of three DNA oligonucleotides containing the universal base analogues 5-nitroindole and 5-nitroindole-3-carboxamide. In all systems studied, both the 5-nitroindole nucleotide and the opposing nucleotide adopt a standard anti conformation and are fully stacked within the DNA duplex. The 5-nitroindole bases do not base pair with the nucleotide opposite them, but intercalate between this base and an adjacent Watson–Crick pair. In spite of their smooth accommodation within the DNA double-helix, the 5-nitroindole-containing duplexes exist as a dynamic mixture of two different stacking configurations exchanging fast on the chemical shift timescale. These configurations depend on the relative intercalating positions of the universal base and the opposing base, and their exchange implies nucleotide opening motions on the millisecond time range. The structure of these nitroindole-containing duplexes explains the mechanism by which these artificial moieties behave as universal bases.

Evolving a Polymerase for Hydrophobic Base Analogues

“…As observed previously,50 both d5NI and d5NIC adopt standard anti conformations (Fig. 11a, b), placing the H2 and H3 protons (purine numbering) in the minor groove of the helix and H6, H8 and H7 (d5NI) or the carboxamide group (d5NIC) in the major groove.…”

“…Cases in point are Pyrene (as its C -nucleoside triphosphate, dPyTP),24,50,51 the triphosphate of d5NI (d5NITP) as well as and a number of other indole derivatives25,52,53. They have been shown to be incorporated with remarkable efficiency and specificity opposite a tetrahydrofuran abasic site analogue (φ).…”

Universal Base Analogues and their Applications to Biotechnology

“…Studies of indole analogues have shown that conversion of the 5-nitro group of 2 to either the 5-amino-or 5-formamido-indole derivatives abolishes its universal base behavior [17]. Alternatively, the addition of hydrogen-bonding groups to 2, to produce 5-nitroindole-3-carboxamide (9), gave no additional advantage in terms of its hybridization properties [18]; moreover, solution studies revealed that the hydrogen-bonding carboxamide group resides in the major groove [19]. Many studies have been conducted to determine the stabilization properties of these universal base analogues.…”

New monolith technology for automated anion-exchange purification of nucleic acids

“…In order to successfully perform purifications on therapeutic ONs, a purification column must resolve isomers arising from these ON modifications. Pellicular anion-exchangers have been shown to resolve many of these including: RNA [8], 2 ,5 -linkages [25,26], phosphoramidate-linked RNA [4,27], phosphorothioate-linked ONs [28,29], DNA containing a variety of modified bases [10], DNA containing intrastrand crosslinks [30], 1-methyl-and 6-methyl-adenine containing oligos [31], cisplatin-modified DNA [32,33], 2 -cyanoethoxymethyl-protected RNA [6], DNA containing a universal base analog (5-nitroindol) [34], and oligonucleotides harboring phosphorothioate diastereoisomers [35]. If these capabilities derive from the pellicular coating, the latexed monolith would constitute a significant advance for purification of clinically important nucleic acids.…”