Michaela Vorlíčková

Circular dichroism and conformational polymorphism of DNA

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 inexpensive. 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.

Circular dichroism and guanine quadruplexes

Circular dichroism (CD) is remarkably sensitive to the conformational states of nucleic acids; therefore, CD spectroscopy has been used to study most features of DNA and RNA structures. Quadruplexes are among the significant noncanonical nucleic acids architectures that have received special attentions recently. This article presents examples on the contribution of CD spectroscopy to our knowledge of quadruplex structures and their polymorphism. The examples were selected to demonstrate the potential of this simple method in the quadruplex field. As CD spectroscopy detects only the global feature of a macromolecule, it should preferably be used in combination with other techniques. On the other hand, CD spectroscopy, often as a pioneering approach, can reveal the formation of particular structural arrangements, to search for the conditions stabilizing the structures, to follow the transitions between various structural states, to explore kinetics of their appearance, to determine thermodynamic parameters and also detect formation of higher order structures. This article aims to show that CD spectroscopy is an important complementary technique to NMR spectroscopy and X-ray diffraction in quadruplex studies.

Guanine tetraplex topology of human telomere DNA is governed by the number of (TTAGGG) repeats

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.

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 G3(TTAG3)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 G3(TTAG3)3 motif. Both G3(TTAG3)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 AG3(TTAG3)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.

Circular dichroism spectroscopy of DNA: from duplexes to quadruplexes.

Nucleic acids bear the genetic information and participate in its expression and evolution during replication, repair, recombination, transcription, and translation. These phenomena are mostly based on recognition of nucleic acids by proteins. The major factor enabling the specific recognition is structure. Circular dichroism (CD) spectroscopy is very useful to study secondary structures of nucleic acids, in general, and DNA, in particular. CD sensitively reflects isomerizations among distinct conformational states. The isomerizations may operate as molecular switches regulating various physiological or pathological processes. Here, we review CD spectra of nucleic acids, beginning with early studies on natural DNA molecules through analyses of synthetic polynucleotides to study of selected genomic fragments.

Salt-induced conformational transition of poly[d(A-T)]·poly[d(A-T)]

Unique chiroptical properties of poly[d(A-T)] X poly[d(A-T)] observed in CsF solutions (Vorlícková et al., 1980) were specified by circular dichroism, ultraviolet light and 31P nuclear magnetic resonance spectroscopy. It was found that up to a 3 M concentration of the salt, caesium cations induced a gradual rearrangement of the polynucleotide double helix during which the phosphodiester bonds in one sequence changed the geometry and the base stacking decreased. At higher CsF concentrations poly[d(A-T)] X poly[d(A-T)] underwent a transition toward a novel conformation which had phosphodiester bonds in both sequences in considerably different non-B-DNA geometries.

Conformational Variability of Poly(dA-dT)·Poly(dA-dT) and Some Other Deoxyribonucleic Acids Includes a Novel Type of Double Helix

The article reviews data indicating that poly(dA-dT).poly(dA-dT) is able of adopting three distinct double helical structures in solution, of which only the A form conforms to classical notions. The other two structures have dinucleotides as double helical repeats. At low salt concentrations poly(dA-dT).poly(dA-dT) adopts a B-type alternating conformation which is exceptionally variable. Its architecture can gradually move in the limits demarcated by the CD spectra with inverted long wavelength CD bands and the 31P NMR spectra with a very low and a 0.6 ppm separation of two resonances. Contrary to Z-DNA, the 31P NMR spectrum of the limiting alternating B conformation of poly(dA-dT).poly(dA-dT) is characterized by an upfield shift of one resonance. We attribute the exceptional conformational flexibility of the alternating B conformation to the unequal tendency of bases in the dA-dT and dT-dA steps to stack. However, by assuming the limiting alternating B conformation, the variability of the synthetic DNA is not exhausted. Specific agents make it isomerize into another conformation by a fast, two-state mechanism, which is reflected by a further deepening of the negative long wavelength CD band and a downfield shift of the 31P NMR resonance of poly(dA-dT).poly(dA-dT) that was constant in the course of the gradual alterations of the alternating B conformation. These changes are, however, qualitatively different from the way poly(dG-dC).poly(dG-dC) behaves in the course of the B-Z isomerization. Poly(dG-dC).poly(dG-dC) displays purine-pyrimidine (dGpdC) resonance in the characteristic downfield position, while the downfield resonance of poly(dA-dT).poly(dA-dT) belongs to the pyrimidine-purine (dTpdA) phosphodiester linkages. Consequently, phosphodiester linkages in the purine-pyrimidine steps play a similar role in the appearance of the Z form to the pyrimidine-purine phosphodiesters in the course of the isomerization of poly(dA-dT).poly(dA-dT). This excludes that the high-salt structures of poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) are members of the same conformational family. We call the high-salt conformation of poly(dA-dT).poly(dA-dT) X-DNA. It furthermore follows from the review that synthetic molecules of DNA with alternating purine-pyrimidine sequences of bases can adopt either the Z form or the X form, or even both, depending on the environmental conditions. This introduces a new dimension into the DNA double helix conformational variability. The possible biological relevance of the X form is suggested by experiments with linear molecules of natural DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Mapping the B-A conformational transition along plasmid DNA.

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.

8-Oxoguanine in a quadruplex of the human telomere DNA sequence

8‐Oxoguanine is a ubiquitous oxidative base lesion. We report here on the effect of this lesion on the structure and stability of quadruplexes formed by the human telomeric DNA sequence 5′‐dG3(TTAG3)3 in NaCl and KCl. CD, PAGE and absorption‐based thermodynamic stability data showed that replacement of any of the tetrad‐forming guanines by 8‐oxoguanine did not hinder the formation of monomolecular, antiparallel quadruplexes in NaCl. The modified quadruplexes were, however, destabilized in both salts, the extent of this depending on the position of the lesion. These results and the results of previous studies on guanine‐to‐adenine exchanges and guanine abasic lesions in the same quadruplex show a noticeable trend: it is not the type of the lesion but the position of the modification that determines the effect on the conformation and stability of the quadruplex. The type of lesion only governs the extent of changes, such as of destabilization. Most sensitive sites were found in the middle tetrad of the three‐tetrad quadruplex, and the smallest alterations were observed if guanines of the terminal tetrad with the diagonal TTA loop were substituted, although even these substitutions brought about unfavorable enthalpic changes. Interestingly, the majority of these base‐modified quadruplexes did not adopt the rearranged folding induced in the unmodified dG3(TTAG3)3 by potassium ions, an observation that could imply biological relevance of the results.

Polymorphism of human telomeric quadruplex structure controlled by DNA concentration: a Raman study

DNA concentration has been recently suggested to be the reason why different arrangements are revealed for K+-stabilized human telomere quadruplexes by experimental methods requiring DNA concentrations differing by orders of magnitude. As Raman spectroscopy can be applied to DNA samples ranging from those accessible by absorption and CD spectroscopies up to extremely concentrated solutions, gels and even crystals; it has been used here to clarify polymorphism of a core human telomeric sequence G3(TTAG3)3 in the presence of K+ and Na+ ions throughout wide range of DNA concentrations. We demonstrate that the K+-structure of G3(TTAG3)3 at low DNA concentration is close to the antiparallel fold of Na+-stabilized quadruplex. On the increase of G3(TTAG3)3 concentration, a gradual transition from antiparallel to intramolecular parallel arrangement was observed, but only for thermodynamically equilibrated K+-stabilized samples. The transition is synergically supported by increased K+ concentration. However, even for extremely high G3(TTAG3)3 and K+ concentrations, an intramolecular antiparallel quadruplex is spontaneously formed from desalted non-quadruplex single-strand after addition of K+ ions. Thermal destabilization or long dwell time are necessary to induce interquadruplex transition. On the contrary, Na+-stabilized G3(TTAG3)3 retains its antiparallel folding regardless of the extremely high DNA and/or Na+ concentrations, thermal destabilization or annealing.