CD of homopolymer DNA-RNA hybrid duplexes and triplexes containing A-T or A-U base pairs.

CD spectra and difference-CD spectra of (a) two DNA X RNA hybrid duplexes (poly[r(A) X d(U)] and poly[r(A) X d(T)]) and (b) three hybrid triplexes (poly-[d(T) X r(A) X d(T)], poly[r(U) X d(A) X r(U)], and poly[r(T) X d(A) X r(T)]) were obtained and compared with CD spectra of six A X U- and A X T-containing duplex and triplex RNAs and DNAs. We found that the CD spectra of the homopolymer duplexes above 260 nm were correlated with the type of base pair present (A-U or A-T) and could be interpreted as the sum of the CD contributions of the single strands plus a contribution due to base pairing. The spectra of the duplexes below 235 nm were related to the polypurine strands present (poly-[r(A)] or poly[d(A)]). We interpret the CD intensity in the intermediate 255-235 nm region of these spectra to be mainly due to stacking of the constituent polypurine strands. Three of the five hybrids (poly[r(A) X d(U)], poly[r(A) X d(T)], and poly[d(T) X r(A) X d(T)]) were found to have heteronomous conformations, while poly[r(U) X d(A) X r(U)] was found to be the most A-like and poly[r(T) X d(A) X r(T)], the least A-like.     

A.U and G.C base pairs in synthetic RNAS have characteristic vacuum UV CD bands.

The vacuum UV CD spectra of GpC, CpG, GpG, poly[r(A)], poly[r(C)], poly[r(U)], poly[r(A-U)], poly[r(G).r(C)], poly[r(A).r(U)], and poly[r(A-U).r(A-U)] were measured down to at least 174 nm. These spectra, together with the published spectra of poly[r(G-C).r(G-C)], CMP, and GMP, were sufficient to estimate the CD changes upon base pairing for four double-stranded RNAs. The vacuum UV CD bands of poly[r(A)], poly[r(C)], and the dinucleotides GpC and CpG were temperature dependent, suggesting that they were due to intrastrand base stacking. The dinucleotide sequence isomers GpC and CpG had very different vacuum UV CD bands, indicating that the sequence can play a role in the vacuum UV CD of single-stranded RNA. The vacuum UV CD bands of the double-stranded (G.C)-containing RNAs, poly[r(G).r(C)] and poly[r(G-C).r(G-C)], were larger than the measured or estimated vacuum UV CD bands of their constituent single-stranded RNAs and were similar in having an exceptionally large positive band at about 185 nm and negative bands near 176 and 209 nm. These similarities were enhanced in difference-CD spectra, obtained by subtracting the CD spectra of the single strands from the CD spectra of the corresponding double strands. The (A.U)-containing double-stranded RNAs poly[r(A).r(U)] and poly[r(A-U).r(A-U)] were similar only in that their vacuum UV CD spectra had a large positive band at 177 nm. The spectrum of poly[r(A).r(U)] had a shoulder at 188 nm and a negative band at 206 nm, whereas the spectrum of poly[r(A-U).r(A-U)] had a positive band at 201 nm. On the other hand, difference spectra of both of the (A.U)-containing polymers had positive bands at about 177 and 201 nm. Thus, the difference-CD spectra revealed CD bands characteristic of A.U and G.C base pairing. (ABSTRACT TRUNCATED AT 250 WORDS)     

The vacuum UV CD spectra of G.G.C triplexes.

Vacuum UV circular dichroism (CD) spectra were measured down to 175 nm for d(C)10, d(G)10, the d(G)10.d(C)10 duplex, and the d(G)10.d(G)10.d(C)10 triplex. A CD difference spectrum was calculated for d(G)10.d(C)10 giving the change in CD induced by forming the duplex from d(G)10 and d(C)10. The d(G)10.d(G)10.d(C)10 CD difference spectrum gave the CD induced by triplex formation from binding of d(G)10 to the d(G)10.d(C)10 duplex. In the near-UV, the d(G)10.d(C)10 and d(G)10.d(G)10.d(C)10 difference spectra resembled the difference spectrum for poly[r(G).r(C)] (Biopolymers 29, 325-333). This similarity may be an indication of similar purine base stacking. The d(G)10.d(G)10.d(C)10 vacuum UV difference spectrum had a negative band at 195 nm and a positive band at 180 nm, making it similar to difference spectra for homopolymer triplexes containing T.A.T and U.A.U triplets (Nucl. Acids Res. 19, 2275-2280). The appearance of these bands in difference spectra should be good indicators of triplex formation. The complementary oligonucleotides c-mycI d(CCCCACCCTCCC) and c-mycII d(GGGAGGGTGGGG) are part of the regulatory sequences of the human c-myc gene. G.G.C rich triplexes formed by binding c-mycII or c-mycIII d(GGGGTGGGTGGG) to the c-mycI.c-mycII duplex had CD difference spectra similar to that of d(G)10.d(G)10.d(C)10 in both the vacuum UV and near UV regions, indicating similar triplet structures.     

The vacuum UV CD bands of repeating DNA sequences are dependent on sequence and conformation

CD spectra were obtained for eight synthetic double-stranded DNA polymers down to at least 175 nm in the vacuum uv. Three sets of sequence isomers were studied: (a) poly[d(A-C).d(G-T)] and poly[d(A-G).d(C-T)], (b) poly[d(A-C-C).d(G-G-T)] and poly[d(A-C-G).d(C-G-T)], and (c) poly[d(A).d(T)], poly[d(A-T).d(A-T)], poly[d(A-A-T).d(A-T-T)], and poly[d(A-A-T-T).d(A-A-T-T)]. There were significant differences in the CD spectra at short wavelengths among each set of sequence isomers. The (G.C)-containing sequences had the largest vacuum uv bands, which were positive and in the wavelength range of 180-191 nm. There were no large negative bands at longer wavelengths, consistent with the polymers all being in right-handed conformations. Among the set of sequences containing only A.T base pairs, poly[d(A).d(T)] had the largest vacuum uv CD band, which was at 190 nm. This CD band was not present in the spectra of the other (A.T)-rich polymers and was absent from two first-neighbor estimations of the poly[d(A).d(T)] spectrum obtained from the other three sequences. We concluded that the sequence dependence of the vacuum uv spectra of the (A.T)-rich polymers was due in part to the fact that poly[d(A).d(T)] exists in a noncanonical B conformation.     

Enhanced stabilization of the triplexes d(C(+)-T)(6):d(A-G)(6);d(C-T)(6), d(T)(21):d(A)(21);d(T)(21) and poly r(U:AU) by water structure-making solutes

A variety of organic cations, cationic lipids, low molecular weight alcohols, sodium dodecylsulfate, trehalose, glycerol, low molecular weight polyethylene glycols, and DMSO were tested for their ability to modulate the stability of the triplexes d(C(+)-T)(6):d(A-G)(6);d(C-T)(6), d(T)(21):d(A)(21);d(T)(21), poly r(U:A U) and their respective core duplexes, d(A-G)(6);d(C-T)(6), d(A)(21);d(T)(21), poly r(A-U). Very substantial enhancement of triplex stability over that in a physiological salt buffer at pH 7 is obtained with different combinations of triplex and high concentrations of these additives, e.g. trimethylammonium chloride and d(C(+)-T)(6):d(A-G)(6);d(C-T)(6); 2-propanol and d(T)(21):d(A)(21);d(T)(21); ethanol and poly r(U:A;U). Triplex formation is even observed with a 1:1 strand mixture of d(A-G)(6) and d(C-T)(6) in the presence of dimethylammonium, tetramethylammonium, and tetraethylammonium-chloride, as well as methanol, ethanol, and 2-propanol. Triplex stability follows the water structure-making ability (and in some cases the duplex unwinding ability) of the organic cations, the low molecular weight alcohols and other neutral organic compounds, whereas water structure-breaking additives decrease triplex stability. These findings are consistent with those reported in the accompanying paper that triplex formation occurs with a net uptake of water. Since the findings suggest that third strand-binding is facilitated by unwinding of the target duplex, it is inferred that triplex formation may be enhanced by nucleic acid binding proteins operating similarly.     

Evidence from CD spectra and melting temperatures for stable Hoogsteen-paired oligomer duplexes derived from DNA and hybrid triplexes.

The pyr*pur.pyr type of nucleic acid triplex has a purine strand that is Hoogsteen-paired with a parallel pyrimidine strand (pyr*pur pair) and that is Watson-Crick-paired with an antiparallel pyrimidine strand (pur.pyr pair). In most cases, the Watson-Crick pair is more stable than the Hoogsteen pair, although stable formation of DNA Hoogsteen-paired duplexes has been reported. Using oligomer triplexes of repeating d(AG)12 and d(CT)12 or r(CU)12 sequences that were 24 nt long, we found that hybrid RNA*DNA as well as DNA*DNA Hoogsteen-paired strands of triplexes can be more stable than the Watson-Crick-paired strands at low pH. The structures and relative stabilities of these duplexes and triplexes were evaluated by circular dichroism (CD) spectroscopy and UV absorption melting studies of triplexes as a function of pH. The CD contributions of Hoogsteen-paired RNA*DNA and DNA*DNA duplexes were found to dominate the CD spectra of the corresponding pyr*pur.pyr triplexes.     

Structure-Function Relationship of Three Triple-Helical Nucleic Acids

Abstract

The nucleic acid triplexes poly d(T)·poly d(A)·poly d(T), poly (U)·poly (A)·poly (U), and poly (I)·poly (A)·poly (I) display a sort of continuity between each other. However, their morphologies present their own individuality which, considering those of their parent duplexes, are quite unexpected. This comparison helps to understand triplex structure-function relationship. While helical parameters are functions of the sugar pucker, low values of WC and Hoogsteen base-pair propellers is commonplace for triplexes and the Hoogsteen base-pair geometry monitors the effects of the interstrand phosphates charge-charge repulsion.

Synopsis

The nucleic acid triplexes poly d(T)·poly d(A)·poly d(T), poly(U)·poly(A)·poly(U), and poly (I)·poly (A)·poly (I) present distinct morphologies. Considering those of their parent duplexes, they are also quite unexpected.     

Spectroscopic studies on the interaction of aristololactam-beta-D-glucoside with DNA and RNA double and triple helices: A comparative study.

The interaction of aristololactam-beta-D-glucoside (ADG), a DNA intercalating alkaloid, with the DNA triplexes, poly(dT). poly(dA)xpoly(dT) and poly(dC).poly(dG)xpoly(dC+), and the RNA triplex poly(rU).poly(rA)xpoly(rU) was investigated by circular dichroic, UV melting profile, spectrophotometric, and spectrofluorimetric techniques. Comparative interaction with the corresponding Watson-Crick duplexes has also been examined under identical experimental conditions. Triplex formation has been confirmed from biphasic thermal melting profiles and analysis of temperature-dependent circular dichroic measurements. The binding of ADG to triplexes and duplexes is characterized by the typical hypochromic and bathochromic effects in the absorption spectrum, quenching of steady-state fluorescence intensity, a decrease in fluorescence quantum yield, an increase or decrease of thermal melting temperatures, and perturbation in the circular dichroic spectrum. Scatchard analysis indicates that ADG binds both to the triplexes and the duplexes in a noncooperative manner. Binding parameters obtained from spectrophotometric measurements are best fit by the neighbor exclusion model. The binding affinity of ADG to the DNA triplexes is substantially stronger than to the RNA triplex. Thermal melting study further indicates that ADG stabilizes the Hoogsteen base-paired third strand of the DNA triplexes whereas it destabilizes the same strand of RNA triplex but stabilizes its Watson-Crick strands. Comparative data reveal that ADG exhibits a stronger binding to the triple helical structures than to the respective double helical structures.     

Structure-function relationship of three triple-helical nucleic acids

The nucleic acid triplexes poly d(T) x poly d(A) x poly d(T), poly (U) x poly (A) x poly (U), and poly (I) x poly (A) x poly (I) display a sort of continuity between each other. However, their morphologies present their own individuality which, considering those of their parent duplexes, are quite unexpected. This comparison helps to understand triplex structure-function relationship. While helical parameters are functions of the sugar pucker, low values of WC and Hoogsteen base-pair propellers is commonplace for triplexes and the Hoogsteen base-pair geometry monitors the effects of the interstrand phosphates charge-charge repulsion.     

Sequence-dependence of the CD of synthetic double-stranded RNAs containing inosinate instead of guanylate subunits

The CD spectra and melting profiles have been measured for nine synthetic double-stranded RNAs containing I · C instead of G · C base pairs: poly[r(I) · r(C)], poly[r(I-C) · r(I-C)], poly[r(A-I-C) · r(I-C-U)], poly[r(A-C) · r(I-U)], poly[r(A-I) · r(C-U)], poly[r(A-C-C) · r(I-I-U)], poly[r(A-A-C) · r(I-U-U)], poly[r(A-C-U) · r(A-I-U)], and poly[r(A-U-C) · r(I-A-U)]. CD spectra have not previously been reported for the latter six of these polymers. The substitution of inosinate for guanylate led to recognizable CD differences, with all but two of the polymers having two resolved positive bands above 230 nm. Also, the I-containing RNAs differed from their G-containing counterparts in the almost complete absence of negative CD bands at long wavelengths and in the reduction of negative CD bands near 210 nm. First-neighbor comparisons showed that the CD spectra of the I-containing RNAs were consistent with the nearest-neighbor sequences of the polymers, as previously shown for G-containing RNAs (D. M. Gray, J.-J. Liu, R. L. Ratliff, and F. S. Allen, Biopolymers (1981) 20 , 1337–1382). Moreover, two of the first-neighbor comparisons involved spectra of poly[r(A) · r(U)] and poly[r(I) · r(C)], polymers known to be in the A family of conformations in fibers (S. Arnott, D. W. L. Hukins, S. D. Dover, W. Fuller, and A. Hodgson, (1973) J. Mol. Biol. 81 , 107–122). Thus, differences in the CD spectra of I- and G-containing RNAs could be simply explained as resulting from differences in the hypoxanthine and guanine chromophores, without invoking differences in conformation. Finally, melting temperatures of the I-containing RNAs were found to vary much less with base composition than do the melting temperatures of G-containing RNAs, since A · U base pairs are closer to I · C than to G · C base pairs in stability.     

Spectroscopic and thermodynamic studies on the binding of sanguinarine and berberine to triple and double helical DNA and RNA structures

A comparative study on the interaction of sanguinarine and berberine with DNA and RNA triplexes and their parent duplexes was performed, by using a combination of spectrophotometric, UV thermal melting, circular dichroic and thermodynamic techniques. Formation of the DNA and RNA triplexes was confirmed from UV-melting and circular dichroic measurements. The interaction process was characterized by increase of thermal melting temperature, perturbation in circular dichroic spectrum and the typical hypochromic and bathochromic effects in the absorption spectrum. Scatchard analysis indicated that both the alkaloids bound to the triplex and duplex structures in a non-cooperative manner and the binding was stronger to triplexes than to parent duplexes. Thermal melting studies further indicated that sanguinarine stabilized the Hoogsteen base paired third strand of both DNA and RNA triplexes more tightly compared to their Watson-Crick strands, while berberine stabilized the third strand only without affecting the Watson-Crick strand. However, sanguinarine stabilized the parent duplexes while no stabilization was observed with berberine under identical conditions. Circular dichroic studies were also consistent with the observation that perturbations of DNA and RNA triplexes were more compared to their parent duplexes in presence of the alkaloids. Thermodynamic data revealed that binding of sanguinarine and berberine to triplexes (T.AxT and U.AxU) and duplexes (A.T and A.U) showed negative enthalpy changes and positive entropy changes but that of sanguinarine to C.GxC(+) triplex and G.C duplex exhibited negative enthalpy and negative entropy changes. Taken together, these results suggest that both sanguinarine and berberine can bind and stabilize the DNA and RNA triplexes more strongly than their respective parent duplexes.     

Sequence and conformational effects on imino proton exchange in A.T- and A.U-containing DNA and RNA duplexes

¹H-nmr relaxation has been used to study the effect of sequence and conformation on imino proton exchange in adenine–thymine (A · T) and adenine–uracil (A · U) containing DNA and RNA duplexes. At low temperature, relaxation is caused by dipolar interactions between the imino and the adenine amino and AH2 protons, and at higher temperature, by exchange with the solvent protons. Although room temperature exchange rates vary between 3 and 12s^?1, the exchange activation energies (E_α) are insensitive to changes in the duplex sequence (alternating vs homopolymer duplexes), the conformation (B-form DNA vs A-form RNA), and the identity of the pyrimidine base (thymine vs uracil). The average value of the activation energy for the five duplexes studied, poly[d(A-T)], poly[d(A) · d(T)], poly[d(A-U)], Poly[d(A) · d(U)], and poly[r(A) · r(U)], was 16.8 ± 1.3 kcal/mol. In addition, we find that the average E_α for the A.T base pairs in a 43-base-pair restriction fragment is 16.4 ± 1.0 kcal/mol. This result is to be contrasted with the observation that the E_α of cytosine-containing duplexes depends on the sequence, conformation, and substituent groups on the purine and pyrimidine bases. Taken together, the data indicate that there is a common low-energy pathway for the escape of the thymine (uracil) imino protons from the double helix. The absolute values of the exchange rates in the simple sequence polymers are typically 3–10 times faster than in DNAs containing both A · T and G · C base pairs.     

Single-Stranded DNA and RNA Targeted Triplex-Formation: UV,CD and Molecular Modeling Studies of Foldback Triplexes Containing Different RNA, 2′-OMe-RNA and DNA Strand Combinations

Abstract

We studied the influence of different 2′-OMe-RNA and DNA strand combinations on single strand targeted foldback triplex formation in the Py.Pu:Py motif using ultraviolet (UV) and circular dichroism (CD) spectroscopy, and molecular modeling. The study of eight combinations of triplexes (D D:D, R* D:D, D D:R*, R* D:R*, D R:D, R* R:D, DR:R*, and R*-R:R*; where the first, middle, and last letters stand for the Hoogsteen Pyrimidine, Watson-Crick [WC] purine and WC pyrimidine strands, respectively, and D, R and R* stand for DNA, RNA and 2′-OMe-RNA strands, respectively) indicate more stable foldback triplex formation with a DNA purine strand than with an RNA purine strand. Of the four possible WC duplexes with RNA/DNA combinations, the duplex with a DNA purine strand and a 2′-O-Me-RNA pyrimidine strand forms the most thermally stable triplex, although its thermal stability is the lowest of all four duplexes. Irrespective of the duplex combination, a 2′-OMe-RNA Hoogsteen pyrimidine strand forms a stable foldback triplex over a DNA Hoogsteen pyrimidine strand confirming the earlier reports with conventional and circular triplexes. The CD studies suggest a B-type conformation for an all DNA homo-foldback triplex (D.D.D), while hetero-foldback triplex spectra suggest intermediate conformation to both Atype and B-type structures. A novel molecular modeling study has been carried out to understand the stereochemical feasibility of all the combinations of foldback triplexes using a geometric approach. The new approach allows use of different combinations of chain geometries depending on the nature of the chain (RNA vs. DNA).     

A comparison of the circular dichroism spectra of synthetic DNA sequences of the homopurine . homopyrimidine and mixed purine- pyrimidine types.

We have obtained the ultraviolet circular dichroism spectra of two repeating trinucleotide DNAs, poly [d(A-G-G).d(C-C-T)] and poly[d(A-A-G).d(C-T-T)], that have all purines on one strand and all pyrimidines on the other. These spectra, together with spectra of other synthetic polymers, can be combined to give 3 first-neighbor calculations of the spectrum of poly[d(A).d(T)] and 2 first-neighbor calculations of the spectrum of poly [d(G).d(C)]. The results show (1) that first-neighbor calculations utilizing only spectra of homopurine.homopyrimidine DNA sequences are no more accurate than are similar calculations that involve spectra of mixed purine-pyrimidine sequences, demonstrating that double-stranded homopurine.homopyrimidine sequences do not obviously belong to a special class of secondary conformations, and (2) that the wavelength region above 250 nm in the CD spectra of synthetic DNAs is least predictable from first-neighbor equations, probably because this region is especially sensitive to sequence-dependent conformational differences.     

Synthetic repeating sequence DNAs containing phosphorothioates: nuclease sensitivity and triplex formation.

More than twenty repeating sequence DNAs containing phosphorothioates were prepared from the appropriate dXTPs with DNA polymerase I. The Tms of the modified DNAs were all lower than the parent polymers. A phosphorothioate group 5' to a pyrimidine gave rise to a large decrease than 5' to a purine, e.g., poly(dA).poly(dT) = 50 degrees; poly(dsA).poly(dT) = 44 degrees; poly(dA).poly(dsT) = 33 degrees; and poly(dsA).poly(dsT) = 26 degrees. The presence of phosphorothioate groups had a dramatic effect on triplex formation; poly[d(TC)].poly[d(sGsA)] spontaneously dismutases to a triplex at pH 8 whereas triplex formation in poly[d(sTsC)].poly[d(GA)] was inhibited. Surprisingly poly(dsG).poly(dC) had a Tm which initially decreased with increasing ionic strength. Resistance to digestion with pancreatic DNAse I did not correlate with phosphorothioate content. Poly[d(AsT)], poly[d(TsC)].poly[d(sGA)] and poly[d(sTG)].poly[d(sCA)] were resistant whereas poly[d(sAT)] and poly[d(sTsTG)].poly[d(CsAsA)] were rapidly degraded. Thus phosphorothioate groups cause small conformational changes and may reveal new families of conformational polymorphisms.     

Structure of the alpha-form of poly[d(A)].poly[d(T)] and related polynucleotide duplexes

The alpha-form of poly[d(A)].poly[d(T)], observed in fibers at high (greater than 80%) relative humidity, is a 10-fold double-helical structure of pitch 3.2 nm. This new X-ray analysis shows that the two strands of the double helix are of the same kind conformationally and both B-like in containing C-2'-endo-puckered deoxyribose rings. Nevertheless, the two strands are different enough for the overall morphology of the duplex to resemble that of the heteromerous model for the drier (beta) form of poly[d(A)].poly[d(T)] in which one strand has C-2'-endo rings and the other C-3'-endo. Since the orientations of the bases in poly[d(A)].poly[d(T)] are persistently different from those of classical B-DNA it is likely that there will be local bending (about 10 degrees) at the junctions between general sequence tracts and the oligo[d(A)].oligo[d(T)] tracts that occur in some native DNAs. The conclusions about the structure of alpha-poly[d(A)].poly[d(T)] are reinforced by independent analyses of similar X-ray diffraction patterns from poly[d(A)].poly[d(U)] and poly[d(A-I)].poly[d(C-T)].     

Intramolecular triplex formation of the purine.purine.pyrimidine type

Six octadecamers with hairpin motifs have been synthesized and investigated for possible intramolecular triplex formation. Electrophoretic, hypochromic, and CD evidence suggest that d(CCCCTTTGGGGTTTGGGG) and d(GGGGTTTGGGGTTTCCCC) can form G.G.C intramolecular triplexes via double hairpin formation in neutral solutions, presumably with the terminal G tract folding back along the groove of the hairpin duplex. In contrast, d(GGGGTTTCCCCTTTGGGG) and the three corresponding 18-mers containing one G and two C tracts each forms a single hairpin duplex with a dangling single strand. The design of the sequences has led to the conclusion that the two G tracts are antiparallel to each other in such a triplex. Magnesium chloride titrations indicate that Mg2+ is not essential for such an intramolecular triplex formation. The main advantage of our constructs when compared to the intermolecular triplex formation is that the shorter triplex stem can be formed in a much lower DNA concentration. The merit of G.G.C triplex, in contrast to that of C+.G.C, lies in the fact that acidic condition is not required in its formation and will, thus, greatly expand our repertoire in the triplex strategy for the recognition and cleavage of duplex DNA. Spectral binding studies with actinomycin D (ACTD) and chromomycin A3 (CHR) as well as fluorescence lifetime measurements with ethidium bromide (EB) suggest that although hairpin duplexes bind these drugs quite well, the intramolecular triplexes bind poorly. Interestingly, the binding densities for the strong-binding hairpins obtained from Scatchard plots are about one ACTD molecule per oligomeric strand, whereas more than two drug molecules are found in the case of CHR, in agreement with the recent NMR studies indicating that CHR binds to DNA in the form of a dimer.     

CD of six different conformational rearrangements of poly[d(A-G).d(C-T)] induced by low pH.

On the basis of circular dichroism (CD) data, we have now identified six different conformational states (other than the duplex) of poly[d(A-G).d(C-T)] at pH values between 8 and 2.5 (at 0.01M Na+; 20 degrees C). Three of these structural rearrangements were observed as the pH was lowered from 8 to 2.5, and three additional rearrangements were observed as the pH was raised from 2.5 back to neutral pH. The major components of the six conformational states were defined using appropriate combinations of the CD spectra of the duplex, triplex, and denatured forms of this polymer, as well as the CD spectra of the individual single strands and their respective acid-induced self-complexes. Our results show that the acid-induced rearrangements of poly[d(A-G).d(C-T)] include not only the poly[d(C+-T).d(A-G).d(C-T)] triplex, but also include the poly[d(C-T)] loop-out structure and a self-complexed form of the poly[d(A-G)] strand that is pH-dependent.     

"Paper-clip" type triple helix formation by 5'-d-(TC)3Ta(CT)3Cb(AG)3 (a and b = 0-4) as a function of loop size with and without the pseudoisocytosine base in the Hoogsteen strand

The formation of a DNA "paper-clip" type triple helix (triplex) with a common sequence 5'-d-(TC)(3)T(a)()(CT)(3)C(b)()(AG)(3) (a and b = 0-4) was studied by UV thermal melting experiments and CD spectra. These DNA oligomers form triplexes and duplexes under slightly acidic and neutral conditions, respectively. The stability of the formed triplexes (at pH 4.5) or duplexes (at pH 7.0 or 8.0) does not vary significantly with the size of the loops (a and b = 1-4). At pH 6.0, the triplex stability is, however, a function of a and b. It is also interesting to note that the oligomer 5'-d-(TC)(3)(CT)(3)(AG)(3) (a and b = 0) forms a stable triplex at pH 4.5 with a slightly lower T(m) value, due to dissociation of a base triad at one end and a distorted base triad at the other, observed by (1)H NMR. Thus, we have here a model system, 5'-d-(TC)(3)T(a)(CT)(3)C(b)(AG)(3), that could form a triplex effectively with (a and b = 1-4) and without (a and b = 0) loops under acidic conditions. In addition, the triplex formation of oligomers with replacement of one, two, or three 2'-deoxycytidine in the Hoogsteen strand by either 2'-deoxypseudoisocytidine (D) or 2'-O-methylpseudoisocytidine (M) was also studied in the sequence 5'-d-(TX)(3)T(2)(CT)(3)C(2)(AG)(3) (where X is C, D, or M). Both CD spectra and UV melting results showed that only D3 [(TX)(3) = (TD)(3)] and M3 [(TX)(3) = (TM)(3)] were able to form the paper-clip structure under both neutral and acidic conditions. This is because the N(3)H of a pseudoisocytosine base can serve as a proton donor without protonation. We hereby proved that the 2'-deoxypseudoisocytidine, similar to 2'-O-methylpseudoisocytidine, could replace 2'-deoxycytidine in the Hoogsteen strand to provide triplex formation at neutral pH.