DSNA
Abstracts
Here we review studies that provided important information about conformational properties of DNA using circular dichroic (CD) spectroscopy. The conformational properties include the B-family of structures, A-form, Z-form, guanine quadruplexes, cytosine quadruplexes, triplexes and other less characterized structures. CD spectroscopy is extremely sensitive and relatively cheap. This fast and simple method can be used at low as well as high DNA concentrations and with short as well as long DNA molecules. The samples can easily be titrated with various agents to cause conformational isomerizations of DNA. The course of detected CD spectral changes makes possible to distinguish between gradual changes within a single DNA conformation and cooperative isomerizations between discrete structural states. It enables measuring kinetics of the appearance of particular conformers and determination of their thermodynamic parameters. In careful hands, CD spectroscopy is a valuable tool for mapping conformational properties of particular DNA molecules. Due to its numerous advantages, CD spectroscopy significantly participated in all basic conformational findings on DNA.
The (CGG) repeats associated with X-chromosome fragility are generally believed to form quadruplexes. This notion has persisted although it had been shown that only very short (CGG)n sequences form quadruplexes and that this quadruplex formation occurs in conditions far from physiological. We have now studied, using CD and absorption spectroscopies, quadruplex formation of (CGG)n (n= 4, 7, 8, or 16) and their analogs interrupted by (AGG) triplets under various solvent conditions. In healthy individuals, (AGG) triplets are interspersed throughout the (CGG) repeat regions and appear to hinder (CGG)n motif expansion. Here we show that (CGG) repeats do not form quadruplexes under physiological conditions in aqueous solution but, interestingly, quadruplexes are readily formed in waterethanol solutions. The presence of (AGG) triplets markedly stabilized quadruplex formation. Quadruplexes may thus hinder rather than support (CGG)n motif expansion.
We have studied the formation and structural properties of quadruplexes of the human telomeric DNA sequence G3(T2AG3)3 and related sequences in which each guanine base was replaced by an adenine base. None of these single base substitutions hindered the formation of antiparallel quadruplexes, as shown by circular dichroism, gel electrophoresis, and UV thermal stability measurements in NaCl solutions. Effect of substitution did differ, however, depending on the position of the substituted base. The A-for-G substitution in the middle quartet of the antiparallel basket scaffold led to the most distorted and least stable structures and these sequences preferred to form bimolecular quadruplexes. Unlike G3(T2AG3)3, no structural transitions were observed for the A-containing analogs of G3(T2AG3)3 when sodium ions were replaced by potassium ions. The basic quadruplex topology remained the same for all sequences studied in both salts. As in vivo misincorporation of A for a G in the telomeric sequence is possible and potassium is a physiological salt, these findings may have biological relevance.
CD spectroscopy, gel electrophoresis and absorption-based thermal stabilitywere used to analyze quadruplex formation of RNA and RNA/DNA hybrid analogs of the deoxyoligonucleotide G4T4G4, which forms a well-characterized basket-type quadruplex. All RNA-containing dodecamers, g4u4g4, G4u4G4 and g4T4g4 (RNA lower-case, DNA capital letters), formed parallel, namely tetramolecular quadruplexes in Na+-containing solutions. The u4 loop forced DNA tetrads into the same conformation as adopted by g4u4g4. In contrast, the T4 loop destabilized the RNA tetrads. Potassium ions markedly stabilized parallel quadruplexes of RNA-containing analogs aswell as their bimolecular folding. In the presence of K+, g4T4g4 formed exclusively bimolecular quadruplexes of both parallel and antiparallel types as indicated by CD. Thus, the T4 loop permits RNA strands to adopt an antiparallel arrangement. These findings may be useful for engineering particular quadruplex foldings in different quadruplex-forming sequences.
Using circular dichroism spectroscopy, gel electrophoresis, and ultraviolet absorption spectroscopy, we have studied quadruplex folding of RNA/DNA analogs of the Oxytricha telomere fragment, G4T4G4, which forms the well-known basket-type, antiparallel quadruplex. We have substituted riboguanines (g) for deoxyriboguanines (G) in the positions G1, G9, G4, and G12; these positions form the terminal tetrads of the G4T4G4 quadruplex and adopt syn, syn, anti, and anti glycosidic geometries, respectively. We show that substitution of a single sugar was able to change the quadruplex topology. With the exception of G4T4G3g, which adopted an antiparallel structure, all the RNA/DNA hybrid analogs formed parallel, bimolecular quadruplexes in concentrated solution at low salt. In dilute solutions (~0.1 mM nucleoside), the RNA/DNA hybrids substituted at positions 4 or 12 adopted antiparallel quadruplexes, which were especially stable in Na+ solutions. The hybrids substituted at positions 1 and 9 preferably formed parallel quadruplexes, which were more stable than the nonmodified G4T4G4 quadruplex in K+ solutions. Substitutions near the 3´end of the molecule affected folding more than substitutions near the 5´end. The ability to control quadruplex folding will allow further studies of biophysical and biological properties of the various folding topologies.
We analysed complete or almost complete nucleotide sequences of the human, chimp, mouse, rat, chicken, dog, and other genomes to find that they contain extremely long (A+T) a (G+C) blocks that do not occur at all in the corresponding randomized sequences. The longest is an (A+T) block containing 1040 consecutive AT pairs that occurs in the 16th human chromosome. The longest human (G+C) block has 261 bp in length. About a half of the longest blocks occur in introns. The (A+T) blocks are discrete units whereas the (G+C) blocks are diffuse. They are embeeded in the genome through connectors longer than 1 kilobase where the (G+C) content gradually decreases to the value of 50%. Remarkably, the (A+T) as well as (G+C) blocks are substantially shorter in the chimp genome. Chicken is characteristic by very long (G+C) blocks that are even longer than in the human genome. Though much shorter, long (G+C) and especially (A+T) blocks occur in lower organisms as well, which means that AT and GC pair clustering is an ancient property that has evolved into large scales in higher eukaryote genomes and the human genome in particular. Very long (A+T) and (G+C) blocks confer specific biophysical properties on DNA that are likely to influence genome folding in cell nuclei and its functional properties.
The (GA)n microsatellite has been known from previous studies to adopt unusual, ordered, cooperatively melting secondary structures in neutral aqueous solutions containing physiological concentrations of salts, at acid pH values or in aqueous ethanol solutions. In order to find more about the primary structure specificity of these structures, we performed parallel comparative studies of related tetranucleotide repeats (GAGC)5, (GAGT)5, and (GACA)5. The general conclusion following from these comparative studies is that the primary structure specificity is fairly high, indicating that not only guanines but also adenines play a significant role in the stabilization of these unusual structures. (GAGC)5 is a hairpin or a duplex depending on DNA concentration. Neither acid pH nor ionic strength or the presence of ethanol changed the secondary structure of (GAGC)5 in a significant way. (GACA)5 forms a weakly stable hairpin in neutral aqueous solutions but forms a duplex at acid pH where cytosine is protonated. (GAGT)5 behaves most similar to (GAGA)5. Salt induces its hairpin- to duplex transition at neutral pH and an isomerization into another, probably parallel stranded, duplex takes place at acid pH. (GAGT)5 is the only of the three present 20-mers that responds to ethanol like (GAGA)5.
DNA guanine quadruplexes are all based on stacks of guanine tetrads, but they can be of many types differing by mutual strand orientation, topology, position and structure of loops, and the number of DNA molecules constituting their structure. Here we have studied a series of nine DNA fragments (G3Xn)3G3, where X=A, C or T, and n=1, 2 or 3, to find how the particular bases and their numbers enable folding of the molecule into quadruplex and what type of quadruplex is formed. We show that any single base between G3 blocks gives rise to only four-molecular parallel-stranded quadruplexes in water solutions. In contrast to previous models, even two Ts in potential loops lead to tetramolecular parallel quadruplexes and only three consecutive Ts lead to an intramolecular quadruplex, which is antiparallel. Adenines make the DNA less prone to quadruplex formation. (G3A2)3G3 folds into an intramolecular antiparallel quadruplex. The same is true with (G3A3)3G3 but only in KCl. In NaCl or LiCl, (G3A3)3G3 prefers to generate homoduplexes. Cytosine still more interferes with the quadruplex, which only is generated by (G3C)3G3, whereas (G3C2)3G3 and (G3C3)3G3 generate hairpins and/or homoduplexes. Ethanol is a more potent DNA guanine quadruplex inducer than are ions in water solutions. It promotes intramolecular folding and parallel orientation of quadruplex strands, which rather corresponds to quadruplex structures observed in crystals.
Polyacrylamide gel electrophoresis is a widely used method to study short DNA fragments in solution. It is, however, a relative method requiring length markers to assess mobility, shape, flexibility and molecularity of the DNA structures of interest. In recent literature we have encountered the use of oligo(dT) fragments as the native PAGE length markers. We show here that this practice is inadequate because oligo(dT) migration is strongly retarded in native polyacrylamide gels. This conclusion is qualitatively true irrespective of the conditions of electrophoresis, oligo(dT) length and gel concentration. Depending on their length, oligo(dT) fragments migrate 2-4 times slower than that would correspond to their nucleotide number. This leads to erroneous conclusions, e.g. determination of the number of associated molecules in guanine quadruplexes or other DNA complexes.
Alternating guanine-adenine strands of DNA are known to self-associate into a parallel-stranded homoduplex at neutral pH, fold into an ordered single-stranded structure at acid pH, and adopt yet another ordered single-stranded conformer in aqueous ethanol. The unusual conformers melt cooperatively and exhibit distinct CD spectra suggestive of a substantial conformational order, but their molecular structures are not known yet. Here we have probed the molecular structures using guanine and adenine analogs lacking the N7 atom, and thus unable of Hoogsteen pairing, or those restrained in the less frequent syn glycosidic orientation. The studies showed that the syn glycosidic orientation of dA residues promoted the neutral homoduplex whereas the syn orientation of dG was incompatible with the homoduplex. In addition, Hoogsteen pairing of dA seemed to be a crucial property of the homoduplex whereas dG did not pair in this way. The situation was the same in both single-stranded conformers with the dG residues. On the other hand, the presence of N7 was important with dA but its syn geometry was not favorable. The present data can be used as restraints to model the unusual molecular structures of the alternating guanine-adenine strands of DNA.
In the literature, the thrombin binding aptamer GGTTGGTGTGGTTGG is generally taken as a prototype of an intramolecular guanine tetraplex of DNA. Our results, however, show that this notion is not true in aqueous solutions. This conclusion is based on a dependence of the CD spectra on aptamer concentration, migration of the aptamer in polyacrylamide gels, and the Ferguson analysis of the gel migration data. The presented data document that the aptamer forms a bimolecular tetraplex. We furthermore show that only an extension of the aptamer by a sequence containing further guanines, or an elongation of loop regions, causes that its tetraplex folding is intramolecular.
Guanine tetraplexes are a biologically relevant alternative of the Watson and Crick duplex of DNA. It is thought that potassium or other cations present in the cavity between consecutive guanine tetrads are an integral part of the tetraplexes. Here we show using CD spectroscopy that ethanol induces the guanine tetraplexes like or even better than potassium cations. We present examples when ethanol stabilizes guanine tetraplexes with fragments when potassium cations fail to do so. Hence besides the A-form or Z-form, ethanol stabilizes another conformation of DNA, i. e. the guanine tetraplexes. We discuss mechanism of the stabilization. Use of ethanol will permit studies of guanine tetraplexes that cannot be induced by potassium cations or other tetraplex-promoting agents. The work demonstrates that still a broader spectrum of nucleotide sequences can fold into guanine tetraplexes than it has so far been thought. Aqueous ethanol may better simulate conditions existing in vivo than the aqueous solutions.
Spectroscopic methods can be divided into three main groups:
- Electronic spectroscopy, including ultraviolet (UV) and visible absorption spectroscopy, circular dichroism (CD) spectroscopy and linear dichroism (LD) spectroscopy.
- Vibrational spectroscopy, including infrared (IR) absorption spectroscopy, Raman spectroscopy and vibrational circular dichroism (VCD) spectroscopy.
- Nuclear magnetic resonance (NMR) spectroscopy.
The basic principles and technical aspects of these methods are given elsewhere in this Encyclopedia. Here we discuss briefly their applications to DNA and RNA samples and the interpretation of such measurements.
Secondary structures of the G-rich strand of human telomere DNA fragments G3 (TTAG3)n, n = 1-16, have been studied by means of circular dichroism spectroscopy and PAGE, in solutions of physiological potassium cation concentrations. It has been found that folding of these fragments into tetraplexes as well as tetraplex thermostabilities and enthalpy values depend on the number of TTAG3 repeats. The suggested topologies include, e.g. antiparallel and parallel bimolecular tetraplexes, an intramolecular antiparallel tetraplex, a tetraplex consisting of three parallel chains and one antiparallel chain, a poorly stable parallel intramolecular tetraplex, and both parallel and antiparallel tetramolecular tetraplexes. G3(TTAG3)3 folds into a single, stable and very compact intramolecular antiparallel tetraplex. With an increasing repeat number, the fragment tetraplexes surprisingly are ever less thermostable and their migration and enthalpy decrease indicate increasing irregularities or domain splitting in their arrangements. Reduced stability and different topology of lengthy telomeric tails could contribute to the stepwise telomere shortening process.
We have used CD spectroscopy, polyacrylamide gel electrophoresis, and UV absorption spectroscopy to study conformational properties of DNA fragments containing (CCA)n and (TGG)n repeats, which are the most length-polymorphic microsatellite sequences of the human genome. The (CCA)n fragments are random single strands at neutral and alkaline pH but they fold into intramolecular intercalated cytosine tetraplexes at mildly acid pH values. More acid values stabilize intermolecular tetraplex formation. The behavior of (TGG)n repeats is more complex. They form hairpins or antiparallel homoduplexes in low salt solutions which, however, are transformed into parallel-stranded guanine tetraplexes at physiological KCI concentrations. Their molecularity depends on the repeat number: (TGG)4 associates into an octameric complex, (TGG)8 forms tetramolecular complexes. (TGG)n with odd repeat numbers (5, 7, and 9) generate bimolecular and tetramolecular tetraplexes. The only (TGG)7 folds into an intramolecular tetraplex at low KCI concentrations, which is antiparallel-stranded. Moreover, the (TGG)n fragments provide various mutually slipped conformers whose population increases with salt concentration and with the increasing repeat number. However, the self-structures of both strands disappear in the presence of the complementary strand because both (TGG)n and (CCA)n prefer to associate into the classical heteroduplex. We suppose that the extreme conformational variability of the DNA strands stands behind the length polymorphism which the (CCA)n/(TGG)n repeats exhibit in the human genome.
A simple method is presented to monitor conformational isomerizations along genomic DNA. We illustrate properties of the method with the B-A conformational transition induced by ethanol in linearized pUC19 plasmid DNA. At various ethanol concentrations, the DNA was irradiated with ultraviolet light, transferred to a restriction endonuclease buffer and the irradiated DNA was cleaved by 17 restriction endonucleases. The irradiation damaged DNA and the damage blocked the restrictase cleavage. The amount of uncleaved, i.e. damaged, DNA depended on the concentration of ethanol in a characteristic S-shape way typical of the cooperative B-A transition. The transition beginning and midpoint were determined for each restriction endonuclease. These data map the B-A transition along the whole polylinker of pUC19 DNA and six evenly distributed recognition sequences within the rest of the plasmid. The transition midpoints fell within the B-A transition region of the plasmid simultaneously determined by CD spectroscopy. The present method complements the previous methods used to study the B-A transition. It can be employed to analyze multikilobase regions of genomic DNA whose restriction endonuclease cleavage fragments can be separated and quantified on agarose gels.
The molecular and crystal structures of three hexahydrobenzo[c]phenanthridine alkaloids (+)-homochelidonine (1), (+)-chelamine (2), and (-)-norchelidonine (3) have been examined by X-ray single crystal diffraction and by CD spectroscopy. All alkaloids 1-3 possess the cis-junction of B/C rings and the C11-hydroxyl in axial position on the half-chair-conformated B ring. The dihedral angles between the aromatic planes A, D are 51.9(1)°, 55.0(1)°/44.8(1), and 22.0(1)° in 1, 2, and 3, respectively. In all compounds 1-3, there is intramolecular H-bonding between C11-OH group and nitrogen. Using a combined approach of X-ray diffraction and CD spectra correlations the absolute configurations of the title alkaloids have been determined as (+)-11S,13R,14S-homochelidonine (1), (+)-11R,12R,13R,14S-chelamine (2), and (-)-11R,13S,14R-norchelidonine (3).
Using CD spectroscopy, UV absorption spectroscopy and polyacrylamide gel electrophoresis, we studied conformational properties of guanine-rich DNA strands of the fragile X chromosome repeats d(GGC)n, d(GCG)n, and d(CGG)n, with n = 2, 4, 8, and 16. These strands are generally considered in the literature to form guanine tetraplexes responsible for the repeat expansion. We, however, show in this paper that the repeats are reluctant to form tetraplexes. At physiological concentrations of either Na+ or K+ ions, the hexamers and dodecamers associate to form homoduplexes and the longer repeats generate homoduplexes and hairpins. The tetraplexes are rarely observed being relatively most stable with d(GGC)n and least stable with d(GCG)n. The tetraplexes are exclusively formed in the presence of K+ ions, at salt concentrations higher than physiological, more easily at higher than physiological temperatures, and they arise with extremely long kinetics (even days). Moreover, the capability to form tetraplexes sharply diminishes with the oligonucleotide length. These facts make the concept of the tetraplex appearance in this motif in vivo very improbable. A hairpin of the fragile X repeats, whose stability increases with the repeat length, rather is the probable structure responsible for the repeat expansion in genomes.
(Guanine+adenine) strands of DNA are known to associate into guanine tetraplexes, homodimerize into parallel or antiparallel duplexes, and fold into a cooperatively melting single strand resembling the protein alpha helix. Using CD spectroscopy and other methods, we studied how this conformational polymorphism depended on the primary structure of DNA. The study showed that d(GGGA)5 and d(GGA)7 associated into homoduplexes at low salt or in the presence of LiCl but were prone to guanine tetraplex formation, especially in the presence of KCl. In addition, they yielded essentially the same CD spectrum in the presence of ethanol as observed with the ordered single strand of d(GA)10. Strands of d(GA)10, d(GGAA)5, d(GAA)7, and d(GAAA)5 associated into homoduplexes in both LiCl and KCl solutions, but not into guanine tetraplexes. d(GAAA)5 and d(GAA)7 further failed to form the single-stranded conformer in aqueous ethanol. Adenine protonation, however, stabilized the single-stranded conformer even in these adenine-rich fragments. The ordered single strands, homoduplexes as well as the guanine tetraplexes, all provided strikingly similar CD spectra, indicating that all of the conformers shared similar base stacking geometries. The increasing adenine content only decreased the conformer thermostability.
We show using polyacrylamide gel electrophoresis that guanine + adenine repeat strands of DNA associate into homoduplexes at neutral pH and moderate ionic strength. The homoduplexes melt in a cooperative way like the Watson-Crick duplex, although the y contain no Watson-Crick base pair. Guanine is absolutely needed for the homoduplex formation and the homoduplex stability increases with the guanine content of the repeat. The present results have implications for the nature of the first replicators, as well as regarding forces stabilizing the duplexes of DNA.
We demonstrate that the characteristic circular dichroism (CD) features of the parallel-stranded DNA tetraplex of d(G4), especially the strong band at 260 nm, are characteristic for the B and A forms of the antiparallel duplex of d(C4G4). Hence, this band evidently originates from intrastrand guanine-guanine stacking, which is therefore very similar in the duplex and tetraplex DNA. In addition, the same type of the CD spectrum is provided by the ordered single strand of d(GA)10. This observation suggests that the ordered single strand of d(GA)10 is stabilized by a core of guanines stacked like in the parallel tetraplex. This view is used to start the modeling of the molecular structure of the, ordered d(GA)10 single strand. Our studies suggest that guanine itself is strong enough to stabilize various secondary structures of DNA, which is a property relevant to thinking about the origin and evolution of molecular replicators.
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 pseudo-achondroplasia. 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.
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 a half of bases whereas the other half stacks 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 a 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) like 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 clashes, no bends and no other significant deviations 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 so easily switch into structure A. 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 the 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).
We show using CD spectroscopy and other methods that addition of dimethylsulphoxide 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. (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 stabilized by dimethylsulphoxide. This conformer might be a DNA analog of the protein alpha helix, which idea has interesting consequences for thinking about the evolution of 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.
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 (CAG)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.
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)n 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 to its duplex with a complementary (GGC)n strand.
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(CnGn) fragments of DNA provide CD spectra suggesting 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 the B-type element. Hence combination of the information provided by CD spectroscopy and NMR spectroscopy, inspires a notion of 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 find out 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 guanines but almost unstacked cytosines. This generated a hole in the double helix center, which is a property characteristic for A-DNA, but absent in 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.
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. Hypothetically 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.
Jovin and coworkers 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 coworkers 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 the 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.
CD spectroscopy was used to analyze conformational properties of a selfcomplementary 54-mer DNA composed of the alternating (CG)3 and (TA)3 hexamers. NaCl induced Z-form in poly(GC), but the 54-mer remained B-form under the same conditions. The B-Z transition was only induced after the addition of NiCl2, but 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). Z-form was also induced in poly(GC) by CsF that switched poly(AT) into 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 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. The 54-mer cooperatively switched into a conformation at the high trifluoroethanol concentrations whose CD spectrum was best consistent with the A-form in the (TA)3 blocks and Z'-form in the (CG)3 blocks. This suggests that the base pairs are tilted in the Z'-form like in the A-form. The present paper 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.
We took 28 fragments of DNA whose crystal structures were known and used CD spectroscopy to search for conditions stabilizing 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 crystallizing 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 crystallization buffers, promoted or suppressed the TFE-induced changes of several fragments to indicate that the crystallization agents can decide which of the possible structures is adopted by the DNA fragment in the crystal.
We studied DNA dodecamers (CAG)4, (CCG)4, (CGG)4 and (CTG)4 by 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.
CD spectroscopy and polyacrylamide gel electrophoresis were used to analyze cooperatively melting conformers of DNA strands containing the GA and TA dinucleotide repeats. The 20-mer (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 the homoduplexes of the alternating GA and TA sequences can coexist in a single DNA molecule. Another 20-mer (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.