Arrangements of human telomere DNA quadruplex in physiologically relevant K+ solutions.

The arrangement of the human telomeric quadruplex in physiologically relevant conditions has not yet been unambiguously determined. Our spectroscopic results suggest that the core quadruplex sequence G(3)(TTAG(3))(3) forms an antiparallel quadruplex of the same basket type in solution containing either K(+) or Na(+) ions. Analogous sequences extended by flanking nucleotides form a mixture of the antiparallel and hybrid (3 + 1) quadruplexes in K(+)-containing solutions. We, however, show that long telomeric DNA behaves in the same way as the basic G(3)(TTAG(3))(3) motif. Both G(3)(TTAG(3))(3) and long telomeric DNA are also able to adopt the (3 + 1) quadruplex structure: Molecular crowding conditions, simulated here by ethanol, induced a slow transition of the K(+)-stabilized quadruplex into the hybrid quadruplex structure and then into a parallel quadruplex arrangement at increased temperatures. Most importantly, we demonstrate that the same transitions can be induced even in aqueous, K(+)-containing solution by increasing the DNA concentration. This is why distinct quadruplex structures were detected for AG(3)(TTAG(3))(3) by X-ray, nuclear magnetic resonance and circular dichrosim spectroscopy: Depending on DNA concentration, the human telomeric DNA can adopt the antiparallel quadruplex, the (3 + 1) structure, or the parallel quadruplex in physiologically relevant concentrations of K(+) ions.

The fragile X chromosome (GCC) repeat folds into a DNA tetraplex at neutral pH.

UV absorption and CD spectroscopy, along with polyacrylamide gel electrophoresis, were used to study conformational properties of DNA fragments containing the trinucleotide repeat (GCC)(n) (n = 4, 8 or 16), whose expansion is correlated with the fragile X chromosome syndrome. We have found that the conformational spectrum of the (GCC)(n) strand is wider than has been shown so far. (GCC)(n) strands adopt the hairpin described in the literature under a wide range of salt concentrations, but only at alkaline (>7.5) pH values. However, at neutral and slightly acid pH (GCC)(4) and (GCC)(8) strands homodimerize. Our data suggest that the homodimer is a bimolecular tetraplex formed by two parallel-oriented hairpins held together by hemi-protonated intermolecular C.C(+) pairs. The (GCC)(16) strand forms the same tetraplex intramolecularly. We further show that below pH 5 (GCC)(n) strands generate intercalated cytosine tetraplexes, whose molecularity depends on DNA strand length. They are tetramolecular with (GCC)(4), bimolecular with (GCC)(8) and monomolecular with (GCC)(16). i-Tetraplex formation is a complex and slow process. The neutral tetraplex, on the other hand, arises with fast kinetics under physiological conditions. Thus it is a conformational alternative of the (GCC)(n) strand duplex with a complementary (GGC)(n) strand.

Conformational properties of DNA fragments containing GAC trinucleotide repeats associated with skeletal displasias.

The human gene for cartilage oligomeric matrix protein contains five tandem repeats of the GAC trinucleotide. Its expansion by one repeat causes multiple epiphyseal dysplasia, while expansion by two repeats or, remarkably, deletion of one repeat causes pseudoachondroplasia. Here we used CD spectroscopy, PAGE and UV absorption spectroscopy to compare conformational properties of the DNA strands containing four, five, six and seven repeats of the GAC trinucleotide. The (GAC)n strands were found to form four distinct ordered conformations, depending on the solution conditions. The first was a foldback, stable at slightly alkaline pH values and low and medium ionic strengths. Increasing salt concentration induced a transition of the foldback into an antiparallel right-handed homoduplex. Both the conformers contained the Watson-Crick G.C pairs while the intervening adenines contributed little to their B-like conformation. Thirdly, the strands associated into a parallel homoduplex stabilized by the hemiprotonated C+.C pairs and by the GpA steps that both favor the parallel DNA strand orientation. The parallel homoduplex was stable even at neutral pH. The fourth conformation was the left-handed Z-DNA, which formed easier with (GAC)n than with (GC)n of comparable length, indicating that the adenines of (GAC)n promoted the left-handed duplex. The paper shows that stability of the above four conformers strongly depends on the GAC repeat number.

(CGA)(4): parallel, anti-parallel, right-handed and left-handed homoduplexes of a trinucleotide repeat DNA.

Conformational properties of microsatellite DNA regions are the probable reason of their expansions in genomes which lead to serious genetic diseases in some cases. Using CD spectroscopy, UV absorption spectroscopy and polyacrylamide gel electrophoresis, we study in this paper conformational properties of (CGA)(4) and compare them with those of (CAG)(4) - a related repeat, connected with Huntington's disease. We show that (CGA)(4) can adopt several distinct conformations in solution. Around neutral pH it forms a parallel-stranded homoduplex containing C(+).C, G.G, and A.A base pairs. Under the same conditions (CAG)(4) forms a hairpin. At slightly alkaline pH values and low ionic strength, (CGA)(4) also folded into a hairpin which transformed into a bimolecular anti-parallel homoduplex at increasing salt concentrations. The duplex easily isomerized into left-handed Z-DNA, implying that the mismatched adenines between G.C pairs facilitate rather than hinder the B-Z transition. No similar changes took place with (CAG)(4). Thus, the conformational repertoire of (CGA)(4) includes parallel, anti-parallel, right-handed, and left-handed homoduplexes. In contrast, (CAG)(4) invariably adopts only a single conformation, namely the very stable hairpin.

Dimethylsulfoxide-stabilized conformer of guanine-adenine repeat strand of DNA.

Circular dichroism spectroscopy and other methods are used to show that the addition of dimethylsulfoxide causes reversible folding of the (GA)(10) strand of DNA into an ordered single-stranded conformer. The ordered conformer melts in a cooperative way and it does not contain protonated adenine. The (TA)(10), (A)(20), and (G)(20) are all unstable in this conformer. To the best of our knowledge, this is the first known ordered conformer of DNA that is stabilized by dimethylsulfoxide. This conformer might be a DNA analog of the protein alpha helix, which is an interesting idea for thinking about the evolution of DNA.

A-like guanine-guanine stacking in the aqueous DNA duplex of d(GGGGCCCC).

We have used CD spectroscopy, NMR spectroscopy and unrestrained molecular dynamics to study conformational properties of a DNA duplex formed by the self-complementary octamer d(GGGGCCCC). Its unusual CD spectrum contains features indicating A-like stacking of half of the bases, whereas the other half stack in a B-like fashion. Unrestrained molecular dynamics simulations converged to a stable B-like double-helix of d(GGGGCCCC). However, the double-helix contained a central hole whose size was half of that occurring in structure A. In the canonical structure B, the hole does not exist at all because the base-pairs cross the double-helix centre. The cytosine bases were stacked in the duplex of d(GGGGCCCC) as in structure B, while stacking of the guanine bases displayed features characteristic for structure A. NMR spectroscopy revealed that the A-like guanine-guanine stacking was accompanied by an increased tendency of the deoxyribose rings attached to the guanine bases to be puckered in an A-like fashion. Otherwise, the duplex of d(GGGGCCCC) showed no clash, no bend and no other significant deviation from structure B. The present analysis demonstrates a remarkable propensity of the guanine runs to stack in an A-like fashion even within the B-DNA framework. This property explains why the oligo(dG). oligo(dC) tracts switch into structure A so easily. Secondly, this property may influence replication, because structure A is replicated more faithfully than structure B. Thirdly, the oligo(dG) runs might have played an important role in early evolution, when DNA took on functions that originally evolved on RNA. Fourthly, the present study extends the vocabulary of DNA secondary structures by the heteronomous duplex of d(GGGGCCCC) in which the B-like strand of oligo(dC) is bound to the A-like strand of oligo(dG).

Conserved guanine-guanine stacking in tetraplex and duplex DNA.

Using a series of suitably chosen oligonucleotides, we demonstrate that the DNA duplex of d(CCCCGGGG) provides an almost identical CD spectrum as the parallel-stranded tetraplex of d(GGGG). The CD spectra are very sensitive to base stacking in DNA so that the above observation indicates that guanine-guanine stacking is essentially the same within the duplex of d(CCCCGGGG) and the tetraplex of d(GGGG). A very similar CD spectrum is also provided by the A-form of d(CCCCGGGG) induced by trifluoroethanol. These results reveal that guanine-guanine stacking is a structural invariant conserved in various nucleic acid conformers. The structural invariance is likely to cohere with evolution of the genetic molecules and be important for fundamental functions, e.g. initiation of transcription.

A nuclease hypersensitive element in the human c-myc promoter adopts several distinct i-tetraplex structures.

Nucleic acid structure-function correlations are pivotal to major biological events like transcription, replication, and recombination. Depending on intracellular conditions in vivo and buffer composition in vitro, DNA appears capable of inexhaustible structure variation. At moderately acidic, or even neutral pH, DNA strands that are rich in cytosine bases can associate both inter- and intramolecularly to form i-tetraplexes. The hemiprotonated cytosine(+)-cytosine base pair constitutes the building block for the formation of i-tetraplexes, and motifs for their formation are frequent in vertebrate genomes. A major control element upstream of the human c-myc gene, which has been shown to interact sequence specifically with several transcription factors, becomes hypersensitive to nucleases upon c-myc expression. The control element is asymmetric inasmuch as that one strand is uncommonly rich in cytosines and exhibits multiple motifs for the formation of i-tetraplexes. To investigate the propensity for their formation we employ circular dichroism (CD) in combination with ultra violet (UV) spectroscopy and native gel electrophoresis. Our results demonstrate the cooperative formation of well-defined i-tetraplex structures. We conclude that i-tetraplex formation occurs in the promoter region of the human c-myc gene in vitro, and discuss implications of possible biological roles for i-tetraplex structures in vivo. Hypothetical formation of intramolecular fold-back i-tetraplexes is important to c-myc transcription, whereas chromosomal translocation events might involve the formation of bimolecular i-tetraplex structures.

An A-type double helix of DNA having B-type puckering of the deoxyribose rings.

DNA usually adopts structure B in aqueous solution, while structure A is preferred in mixtures of trifluoroethanol (TFE) with water. However, the octamer d(CCCCGGGG) and other d(C(n)G(n)) fragments of DNA provide CD spectra that suggest that the base-pairs are stacked in an A-like fashion even in aqueous solution. Yet, d(CCCCGGGG) undergoes a cooperative TFE-induced transition into structure A, indicating that an important part of the aqueous duplex retains structure B. NMR spectroscopy shows that puckering of the deoxyribose rings is of the B-type. Hence, combination of the information provided by CD spectroscopy and NMR spectroscopy suggests an unprecedented double helix of DNA in which A-like base stacking is combined with B-type puckering of the deoxyribose rings. In order to determine whether this combination is possible, we used molecular dynamics to simulate the duplex of d(CCCCGGGG). Remarkably, the simulations, completely unrestrained by the experimental data, provided a very stable double helix of DNA, exhibiting just the intermediate B/A features described above. The double helix contained well-stacked guanine bases but almost unstacked cytosine bases. This generated a hole in the double helix center, which is a property characteristic for A-DNA, but absent from B-DNA. The minor groove was narrow at the double helix ends but wide at the central CG step where the Watson-Crick base-pairs were buckled in opposite directions. The base-pairs stacked tightly at the ends but stacking was loose in the duplex center. The present double helix, in which A-like base stacking is combined with B-type sugar puckering, is relevant to replication and transcription because both of these phenomena involve a local B-to-A transition.

Circular dichroism spectroscopy analysis of conformational transitions of a 54 base pair DNA duplex composed of alternating CGCGCG and TATATA blocks.

CD spectroscopy was used to analyze conformational properties of a self-complementary 54-mer DNA composed of alternating (CG)3 and (TA)3 hexamers. NaCl induced the Z-form in poly(GC), but the 54-mer remained the B-form under the same conditions. The B-Z transition was induced only after the addition of NiCl2. However, the Z-form was adopted by the whole molecule, not by the (CG)3 blocks alone. Two orders of magnitude higher concentrations of NiCl2 were required to induce the Z-form in poly(AT). The Z-form was also induced in poly(GC) by CsF that switched poly(AT) into the X-form, which seems to be a solution counterpart of D-DNA. Under these conditions the CD spectrum of the 54-mer was consistent with the (TA)3 blocks being in the X-form and the (CG)3 blocks in the B-form. At high concentrations of ethanol or trifluoroethanol, poly(AT) was an A-form, while poly(GC) adopted either Z-form, A-form or Z'-form. At the high trifluoroethanol concentrations the 54-mer cooperatively switched into a conformation whose CD spectrum was most consistent with the A-form in the (TA)3 blocks and the Z'-form in the (CG)3 blocks. This suggests that the base pairs are tilted in the Z'-form as in the A-form. The present article illustrates that CD spectroscopy can provide interesting pieces of information about conformational isomerizations and coexistence of multiple conformations in DNA molecules containing blocks of different simple sequence repeats.

Aqueous trifluorethanol solutions simulate the environment of DNA in the crystalline state.

We took 28 fragments of DNA whose crystal structures were known and used CD spectroscopy to search for conditions stabilising the crystal structures in solution. All 28 fragments switched into their crystal structures in 60-80% aqueous trifluorethanol (TFE) to indicate that the crystals affected the conformation of DNA like the concentrated TFE. The fragments crystallising in the B-form also underwent cooperative TFE-induced changes that took place within the wide family of B-form structures, suggesting that the aqueous and crystal B-forms differed as well. Spermine and magnesium or calcium cations, which were contained in the crystallisation buffers, promoted or suppressed the TFE-induced changes of several fragments to indicate that the crystallisation agents can decide which of the possible structures is adopted by the DNA fragment in the crystal.

Dimerization of the guanine-adenine repeat strands of DNA.

Jovin and co-workers have demonstrated that DNA strands containing guanine-adenine repeats generate a parallel-stranded homoduplex. Here we propose that the homoduplex is a dimer of the ordered single strand discovered by Fresco and co-workers at acid pH. The Fresco single strand is shown here to be stabilized in aqueous ethanol where adenine is not protonated. Furthermore, we demonstrate that the strands dimerize at higher salt concentrations without significantly changing their conformation, so that the dimerization is non-cooperative. Hence, the Jovin homoduplex can form through a non-cooperative dimerization of two cooperatively melting single strands. The available data indicate that the guanines stabilize the Fresco single strand whereas the adenines cause dimerization owing to their known intercalation or clustering tendency. The guanine-adenine repeat dimer seems to be a DNA analog of the leucine zipper causing dimerization of proteins.

Conformational properties of DNA dodecamers containing four tandem repeats of the CNG triplets.

We studied DNA dodecamers (CAG)4, (CCG)4, (CGG)4 and (CTG)4by CD spectroscopy and polyacrylamide gel electrophoresis. Each dodecamer adopted several ordered conformers which denatured in a cooperative way. Stability of the conformers depended on the dodecamer concentration, ionic strength, temperature and pH. The dodecamers, having a pyrimidine base in the triplet center, generated foldbacks at low ionic strength whose stem conformations were governed by the GC pairs. At high salt, (CCG)4 isomerized into a peculiar association of two strands. The association was also promoted by high oligonucleotide concentrations. No similar behavior was exhibited by (CTG)4. At low salt, (CGG)4 coexisted in two bimolecular conformers whose populations were strongly dependent on the ionic strength. In addition, (CGG)4 associated into a tetraplex at acidic pH. A tetraplex was even observed at neutral pH if the (CGG)4 concentration was sufficiently high. (CAG)4 was very stable in a monomolecular conformer similar to the known extremely stable foldback of the (GCGAAGC) heptamer. Nevertheless, even this very stable conformer disappeared if (CTG)4 was added to the solution of (CAG)4. Association of the complementary strands was also strongly preferred to the particular strand conformations by the other couple, (CCG)4 and (CGG)4.

Conformational properties of DNA strands containing guanine-adenine and thymine-adenine repeats.

CD spectroscopy and PAGE were used to cooperatively analyze melting conformers of DNA strands containing GA and TA dinucleotide repeats. The 20mer (GA)10 formed a homoduplex in neutral solutions containing physiological concentrations of salts and this homoduplex was not destabilized even in the terminal (GA)3 hexamers of (GA)3(TA)4(GA)3, although the central (TA)4 portion of this oligonucleotide preserved the conformation adopted by (TA)10. This observation demonstrates that homoduplexes of alternating GA and TA sequences can co-exist in a single DNA molecule. Another 20mer, (GATA)5, adopted as a whole either the AT duplex, like (TA)10, or the GA duplex, like (GA)10, and switched between them reversibly. The concentration of salt controlled the conformational switching. Hence, guanine and thymine share significant properties regarding complementarity to adenine, while the TA and GA sequences can stack in at least two mutually compatible ways within the DNA duplexes analyzed here. These properties extend our knowledge of non-canonical structures of DNA.

Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine.

Using CD spectroscopy, guanine tetraplex formation was studied with short DNA fragments in which cytosine residues were systematically added to runs of guanine either at the 5' or 3' ends. Potassium cations induced the G-tetraplex more easily with fragments having the guanine run at the 5' end, which is just an opposite tendency to what was reported for (G+T) oligonucleotides. However, the present (G+C) fragments simultaneously adopted other conformers that complicated the analysis. We demonstrate that repeated freezing/thawing, performed at low ionic strength, is a suitable method to exclusively stabilize the tetraplex in the (G+C) DNA fragments. In contrast to KCl, the repeated freeze/thaw cycles better stabilized the tetraplex with fragments having the guanine run on the 3' end. The tendency of guanine blocks to generate the tetraplex destabilized the d(G5).d(C5) duplex whose strands dissociated, giving rise to a stable tetraplex of (dG5) and single-stranded (dC5). In contrast to d(G3C3) and d(G5C5), repeated freezing/thawing induced the tetraplex even with the self-complementary d(C3G3) or d(C5G5); hence the latter oligonucleotides preferred the tetraplex to the apparently very stable duplex. The tetraplexes only included guanine blocks while the 5' end cytosines interfered neither with the tetraplex formation nor the tetraplex structure.

Comparison of the solution and crystal conformations of (G + C)-rich fragments of DNA.

DNA fragments crystallize in an unpredictable manner, and relationships between their crystal and solution conformations still are not known. We have studied, using circular dichroism spectroscopy, solution conformations of (G + C)-rich DNA fragments, the crystal structures of which were solved in the laboratory of one of the present authors. In aqueous trifluorethanol (TFE) solutions, all of the examined oligonucleotides adopted the same type of double helix as in the crystal. Specifically, the dodecamer d(CCCCCGCGGGGG) crystalized as A-DNA and isomerized into A-DNA at high TFE concentrations. On the other hand, the hexamer d(CCGCGG) crystallized in Z-form containing tilted base pairs, and high TFE concentrations cooperatively transformed it into the same Z-form as adopted by the RNA hexamer r(CGCGCG), although d(CCGCGG) could isomerize into Z-DNA in the NaCl + NiCl2) aqueous solution. The fragments crystallizing as B-DNA remained B-DNA, regardless of the solution conditions, unless they denatured or aggregated. Effects on the oligonucleotide conformation of 2-methyl-2,4-pentanediol and other crystallization agents were also studied. 2-Methyl-2,4-pentanediol induced the same conformational transitions as TFE but, in addition, caused an oligonucleotide condensation that was also promoted by the other crystallization agents. The present results indicate that the crystal double helices of DNA are stable in aqueous TFE rather than aqueous solution.

The unusual X-form DNA in oligodeoxynucleotides: dependence of stability on the base sequence and length.

X-form is an unusual double helix of DNA adopted by poly(dA-dT) or (dT-dA)4 at high concentrations of CsF. On the other hand, poly(dA), poly(dT), (dA-dT)4 and most other DNAs do not adopt this conformer. Here we demonstrate that the X-form is strongly destabilized by GC pairs or even minute perturbations of the alternating pyrimidinepurine sequence. For example, the 30-mer d(TATAAT)5, containing five tandem repeats of the Pribnow box, fails to isomerize into the X-form. After (dT-dA)4, the 16-mer (dT-dA)8 is shown to be the second most predisposed oligodeoxynucleotide in the (dT-dA)n series to isomerize into the X-form while the duplex lengths corresponding to n = 3,5,6,7,9,12 and 20 make the X-form unstable even in the strictly alternating (dT-dA)n sequence. Consequently, the (dT-dA)n duplex length is also a crucial factor of the X-form stability on the oligodeoxynucleotide level. We discuss a possibility that the X-form is a solution counterpart of the D-form adopted in dehydrated poly(dA-dT) fibers because properties of these two conformers are remarkably similar in many respects.

Unusual contribution of 2-aminoadenine to the thermostability of DNA.

The poly(dA-dU) and poly(dI-dC) duplexes have very similar thermostabilities (Tm). This similarity extends also to the pyrimidine 5-methyl group-containing poly(dA-dT) and poly(dI-m5dC). The differences between chemical structures of the A:U and I:C or the A:T and I:m5C base-pairs seem to be unimportant for the thermostability of the DNA. However, on the insertion of an amino group into position 2 of the purines the similarities disappear. Thermostabilities of poly(n2dA-dU) and poly(dG-dC) as well as the poly(n2dA-dT) and poly(dG-m5dC) are radically different. This is also the case with their other 5-substituted pyrimidine-containing derivatives, the 5-ethyl, 5-n-butyl and 5-bromo analogues. The G:C-based polynucleotides are more stable by an average of 40 degrees C than the n2A.U-based ones. Poly(dA,n2dA-dT)-s containing various proportions of A and n2A as well as the natural DNA of S-2L cyanophage that contains n2A bases instead of A were also studied. It was found that dependence of Tm on the n2A-content was non-linear and that the lower Tm is not the consequence of a particular nucleotide sequence. The possible structural reasons for the lower thermostabilization of these B-DNAs by the n2A:T base-pair as compared to the G:C are discussed.

Divalent zinc cations induce the formation of two distinct homoduplexes of a d(GA)20 DNA sequence.

Homopurine DNA sequences are highly structurally polymorphic. In particular, d(GA)n DNA sequences are known to be capable of forming intramolecular foldbacks, bimolecular homoduplexes, and tetrastranded complexes. Counterions play a determinant role on the equilibria between the different structural conformers of d(GA)n sequences. In this paper, the effect of divalent zinc cations on the structure of a d(GA)20 oligonucleotide has been analyzed by CD spectroscopy and polyacrylamide gel electrophoresis. Depending on the precise experimental conditions at which zinc is added, two distinct conformations of the d(GA)20 oligonucleotide are stabilized. At neutral pH in the absence of zinc, d(GA)20 is partially organized into intramolecular foldbacks and bimolecular homoduplexes [Casasnovas et al. (1993) J. Mol. Biol. 233, 671-681]. Under these conditions, addition of zinc results in the stabilization of the bimolecular homoduplex which is nonspecific for zinc since it is also stabilized by divalent magnesium cations, increasing ionic strength, or decreasing pH. Its CD spectrum is identical to that reported earlier for parallel-stranded d(GA)n homoduplexes [Rippe et al. (1992) EMBO J. 11, 3777-3786]. On the other hand, if zinc is added under conditions where the d(GA)20 oligonucleotide is exclusively single-stranded, a different bimolecular homoduplex appears which is only observed in the presence of zinc. The zinc-specific duplex melts cooperatively, and, in contrast to the nonspecific duplex, its thermostability is high. Transition from the nonspecific to the zinc-specific duplex is observed at high zinc concentrations or at high temperatures. The transition is cooperative. These results are discussed in the context of the specific cation effects on the formation of intramolecular R.R.Y triplexes at d(GA.TC)n DNA sequences.