[19] Circular dichroism spectroscopy of DNA

The structure of a stable intermediate in the A ↔ B DNA helix transition

“…The trapping of an A͞B DNA intermediate in the G 3 C 3 crystal challenges results from CD spectroscopic experiments that suggest that the A 7 B transition occurs abruptly between two discrete states (11,12). However, CD absorbance may be insensitive to the presence of AB intermediates containing base-pair inclination angles similar to those of B DNA, because a major component of the CD signal depends on base-pair inclination (27). CD measurements may be detecting only one aspect of the B-to-A transition.…”

Aggregated DNA in ethanol solution

“…Special precautions must be taken if DNA is to be studied in dissolved form up to 75% concentration [15]. At higher ethanol concentrations several conformational transitions, like B-to-A [16,17,18] and B-to-C [19] were observed for such dissolved DNA. Until recently studies of the heat-induced helix-to-coil transition at higher ethanol concentrations have been performed only on dissolved DNA.…”

Specific metal-ion binding sites in a model of the P4-P6 triple-helical domain of a group I intron

“…Here we have studied the formation of the core triple helix motif in a two-component model system+ We have used difference circular dichroism (CD) spectroscopy in a novel approach to probe structurally important magnesium-ion binding sites within the motif+ This structural approach is in contrast to many other phosphorothioate experiments, which use activity as an assay (Dahm & Uhlenbeck, 1991;Chowrira & Burke, 1992;Christian & Yarus, 1993;Piccirilli et al+, 1993;Scott & Uhlenbeck, 1999)+ Difference CD spectroscopy emphasizes the spectral differences of an associated system as compared to its nonassociated state (Johnson, 1985;Gray et al+, 1992)+ It can also be used to compare a modified system to its unmodified counterpart+ By combining these two approaches, we were able to separate the effects of the metal-ion switch (Mg 2ϩ r Mn 2ϩ ) on the CD spectra from the effects of a true structural rescue by Mn 2ϩ + Our model system consists of a 7-mer ribonucleotide (7-mer RNA) and a 23-mer mixed ribo-deoxyribo oligonucleotide (23-mer RNA) (Fig+ 1B,C)+ These have been shown by us, in earlier studies using CD and FTIR spectroscopy (Sarkar et al+, 1996(Sarkar et al+, , 1997, to associate in the presence of Mg 2ϩ , modeling the interactions in the P4-P6 triple-helical domain of a self-splicing group I intron from bacteriophage T4 nrdB pre-mRNA (Fig+ 1A), which has a core sequence analogous to the Tetrahymena ribozyme (Sjöberg et al+, 1986)+ The assembly process of this two-strand model system may in certain aspects better resemble the native folding as compared to a single-stranded model, as it is not constrained by the covalent connectivity in a short strand+ The model has previously been shown to be sensitive to effects of base substitutions (Sarkar et al+, 1996) and 29-OH substitutions (Sarkar et al+, 1997) on triple helix formation+ In the present study, the stereospecific effects of Rp and Sp sulfur substitutions at the 59 end of nucleotides U452 and U453 are evaluated+ U452 and U453 are positioned at the junction of helix P6 and helix P4 in the triple-helical model system (Fig+ 1C), and correspond to nucleotides U258 and U259 of the Tetrahymena ribozyme+…”

“…phage T4 nrdB (nt 449-455 in the original sequence), and a 23-mer RNA having the same base sequence as the J3/4-P4 (59-end bases of P4, nt 42-51), followed by three deoxythymidines (dT 3 ) replacing the P5 loop, and the P4-P6 (39-end bases of P4 and 59-end bases of P6, respectively, nt 70-79)+ The P5 loop in the original sequence is replaced by dT 3 , as they form a stable hairpin loop that should not be involved in RNA tertiary interactions (Sarkar et al+, 1996) (Sarkar et al+, 1996), having the overall features characteristic of an A-helix, with a large positive band around 250-300 nm and a narrower but negative band positioned around 210 nm (Johnson, 1985;Gray et al+, 1992)+ The singlestranded nonsubstituted 7-mer RNA (7RNA ns) and phosphorothioate substituted 7-mer RNAs (U452 Rp, U452 Sp, U453 Rp, and U453 Sp) all showed CD spectra characteristic of non-base-pairing oligonucleotides (Johnson, 1985; data not shown)+ Triple helix formation of 23-mer RNA and nonsubstituted 7-mer RNA CD spectroscopy is very sensitive to conformational changes of nucleic acids+ If a triple helix is formed in a mixture of 23-mer RNA and one of the single-stranded 7-mer RNAs, the resulting CD spectrum differs from that of a nonassociated system (Gray et al+, 1992)+ The spectrum of nonassociated 23-mer RNA and 7-mer RNA at a specific divalent metal-ion concentration is equal to the sum of the individual spectra of 23-mer RNA and 7-mer RNA recorded under identical salt conditions+ Utilizing this fact, we recorded a set of CD spectra of 23-mer RNA, 7-mer RNA, and a 1:1 mixture (in single strands) of 23-mer RNA and 7-mer RNA+ Each set was recorded at five different ionic strengths (0, 5, 50, 100, and 200 mM) and in three different molar ratios (1:0, 3:1, and 0:1) of Mg 2ϩ :Mn 2ϩ + We added the appropri- (Michel & Westhof, 1990; in the shallow groove of helix P6 and in the deep groove of helix P4 are indicated by dotted bonds+ The 2 nt in question for phosphorothioate substitutions (U452 and U453) are marked with slanting lines and dots, respectively+ The substitutions are made 59 to U452 and U453, as shown by arrows+ ately weighted spectra of 23-mer RNA and 7-mer RNA (corresponding to a nonassociated triple-helical system), and compared this calculated spectrum to the one observed for the 1:1 mixture of 23-mer RNA and 7-mer RNA+ If no association takes place in the 1:1 mixture, the observed and calculated spectra overlap as for the wild-type system (23-mer RNA and nonsubstituted 7RNA ns) in 0 mM Mg 2ϩ (Fig+ 2A), and the difference spectrum (observed Ϫ calculated) follows the baseline (Fig+ 2C)+ When association takes place, the observed and calculated spectra differ, as for the wild-type system in 100 mM Mg 2ϩ (Fig+ 2B)+ The observed spectrum of the 1:1 mixture of 23-mer RNA and 7RNA ns at 100 mM Mg 2ϩ has a more intense and slightly narrowed positive band around 250-300 nm than the calculated (nonassociated) spectrum recorded under identical salt conditions+ As a result, a 269-nm positive peak and two zero-crossover points around 263 and 278 nm arise in the difference spectrum, due to the more intense and slightly narrowed 250-300 nm band of the associated system+ The enhancement of the spectral features characteristic of triple helix formation upon raising the Mg 2ϩ concentration reflects an increased degree of association between the RNA oligomers (Fig+ 2C)+ The isolated triple-helical model system requires high divalent ionic strengths to associat...…”