DNA minicircles capable of forming a variety of non-canonical structural motifs.

Although more than 10% of the human genome has the potential to fold into non-B DNA, the formation of non-canonical structural motifs as part of long dsDNA chains are usually considered as unfavorable from a thermodynamic point of view. However, recent experiments have confirmed that non-canonical motifs do exist and are non-randomly distributed in genomic DNA. This distribution is highly dependent not only on the DNA sequence but also on various other factors such as environmental conditions, DNA topology and the expression of specific cellular factors in different cell types. In this study, we describe a new strategy used in the preparation of DNA minicircles containing different non-canonical motifs which arise as a result of imperfect base pairing between complementary strands. The approach exploits the fact that imperfections in the pairing of complementary strands thermodynamically weaken the dsDNA structure at the expense of enhancing the formation of non-canonical motifs. In this study, a completely different concept of stable integration of a non-canonical motif into dsDNA is presented. Our approach allows the integration of various types of non-canonical motifs into the dsDNA structure such as hairpin, cruciform, G-quadruplex and i-motif forms but also combinations of these forms. Small DNA minicircles have recently become the subject of considerable interest in both fundamental research and in terms of their potential therapeutic applications.

G-QINDER Tool: Bioinformatically Predicted Formation of Different Four-Stranded DNA Motifs from (GT)n and (GA)n Repeats.

The recently introduced semi-orthogonal system of nucleic acid imaging offers a greatly improved method of identifying DNA sequences that are capable of adopting noncanonical structures. This paper uses our newly developed G-QINDER tool to identify specific repeat sequences that adopt unique structural motifs in DNA: TG and AG repeats. The structures were found to adopt a left-handed G-quadruplex form under extreme crowding conditions and a unique tetrahelical motif under certain other conditions. The tetrahelical structure likely consists of stacked AGAG-tetrads but, unlike G-quadruplexes, their stability does not appear to be dependent on the type of monovalent cation present. The occurrence of TG and AG repeats in genomes is not rare, and they are also found frequently in the regulatory regions of nucleic acids, so it is reasonable to assume that putative structural motifs, like other noncanonical forms, could play an important regulatory role in cells. This hypothesis is supported by the structural stability of the AGAG motif; its unfolding can occur even at physiological temperatures since the melting temperature is primarily dependent on the number of AG repeats in the sequence.

Hidden Information Revealed Using the Orthogonal System of Nucleic Acids.

In this study, the organization of genetic information in nucleic acids is defined using a novel orthogonal representation. Clearly defined base pairing in DNA allows the linear base chain and sequence to be mathematically transformed into an orthogonal representation where the G-C and A-T pairs are displayed in different planes that are perpendicular to each other. This form of base allocation enables the evaluation of any nucleic acid and predicts the likelihood of a particular region to form non-canonical motifs. The G4Hunter algorithm is currently a popular method of identifying G-quadruplex forming sequences in nucleic acids, and offers promising scores despite its lack of a substantial rational basis. The orthogonal representation described here is an effort to address this incongruity. In addition, the orthogonal display facilitates the search for other sequences that are capable of adopting non-canonical motifs, such as direct and palindromic repeats. The technique can also be used for various RNAs, including any aptamers. This powerful tool based on an orthogonal system offers considerable potential for a wide range of applications.

Rhodamine 6G-Ligand Influencing G-Quadruplex Stability and Topology.

The involvement of G-quadruplex (G4) structures in nucleic acids in various molecular processes in cells such as replication, gene-pausing, the expression of crucial cancer-related genes and DNA damage repair is well known. The compounds targeting G4 usually bind directly to the G4 structure, but some ligands can also facilitate the G4 folding of unfolded G-rich sequences and stabilize them even without the presence of monovalent ions such as sodium or potassium. Interestingly, some G4-ligand complexes can show a clear induced CD signal, a feature which is indirect proof of the ligand interaction. Based on the dichroic spectral profile it is not only possible to confirm the presence of a G4 structure but also to determine its topology. In this study we examine the potential of the commercially available Rhodamine 6G (RhG) as a G4 ligand. RhG tends to convert antiparallel G4 structures to parallel forms in a manner similar to that of Thiazole Orange. Our results confirm the very high selectivity of this ligand to the G4 structure. Moreover, the parallel topology of G4 can be verified unambiguously based on the specific induced CD profile of the G4-RhG complex. This feature has been verified on more than 50 different DNA sequences forming various non-canonical structural motifs.

The effect of single nucleotide polymorphisms in G-rich regions of high-risk human papillomaviruses on structural diversity of DNA.

BACKGROUND: Infection with high-risk human papillomaviruses (HPVs) can lead to development of cancer of the head and neck and anogenital regions. G-rich sequences found in genomes of high-risk HPVs can fold into non-canonical secondary structures that could serve as 3D motifs distinct from double-stranded DNA and present recognition sites for ligands and opportunity for gene expression modulation. METHODS: Combination of UV, CD and NMR spectroscopy and PAGE electrophoresis were used as they offer complementary insights into structural changes of G-rich oligonucleotides. RESULTS: G-rich region of HPV16 is shown to preferentially form hairpin structures, while regions of HPV18, HPV52 and HPV58 fold into four-stranded DNA structures called G-quadruplexes. Single nucleotide polymorphisms found in G-rich sequences have been found to promote formation of hairpin structures of HPV16 and have affected number of species formed in G-rich region of HPV52, whereas they have exhibited minimal effect on the formation of HPV18 and HPV58 G-quadruplex structures. These structural changes were reflected in differences in apparent thermal stabilities. CONCLUSIONS: Potential of G-rich sequences as drug targets was evaluated based on the results of the current study. HPV16 and HPV18 are considered less appropriate targets due to several single nucleotide polymorphisms and low stability, respectively. On the other hand, HPV52 and HPV58 could be used for small-molecule mediated stabilization. GENERAL SIGNIFICANCE: G-rich sequences occurring in high-risk HPVs can fold into hairpin and G-quadruplex structures that could be potentially utilized as drug targets. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Ebola virus derived G-quadruplexes: Thiazole orange interaction.

The Ebola and Marburg viruses are some of the deadliest viruses in the world. In this study a series of G-rich DNA sequences derived from these types of viruses which possess the potential to form G-quadruplex structures are analyzed. A set of DNA oligonucleotides derived from original viral isolates was used as a representative modeling sequence with which to demonstrate the influence of thiazole orange on circular dichroism (CD) spectral profiles. The results show the unique profile of the induced CD (ICD) signal in the visible region caused by interactions between the ligand and G-quadruplexes. This ligand was found to stabilize the G-quadruplex structure and can also induce topological changes and facilitate G-quadruplex multimerization. Thus, the ICD signatures can be used to determine whether specific unknown sequences can form G-quadruplex motifs. The viral sequences were analyzed using standard spectral and electrophoretic methods. In addition, the ability to target G-quadruplexes located in filoviruses offers researchers attractive therapeutic targets which would be of particular use in the development of novel antiviral therapies. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Telomeric G-Quadruplexes: From Human to Tetrahymena Repeats.

The human telomeric and protozoal telomeric sequences differ only in one purine base in their repeats; TTAGGG in telomeric sequences; and TTGGGG in protozoal sequences. In this study, the relationship between G-quadruplexes formed from these repeats and their derivatives is analyzed and compared. The human telomeric DNA sequence G3(T2AG3)3 and related sequences in which each adenine base has been systematically replaced by a guanine were investigated; the result is Tetrahymena repeats. The substitution does not affect the formation of G-quadruplexes but may cause differences in topology. The results also show that the stability of the substituted derivatives increased in sequences with greater number of substitutions. In addition, most of the sequences containing imperfections in repeats which were analyzed in this study also occur in human and Tetrahymena genomes. Generally, the presence of G-quadruplex structures in any organism is a source of limitations during the life cycle. Therefore, a fuller understanding of the influence of base substitution on the structural variability of G-quadruplexes would be of considerable scientific value.

Putative HIV and SIV G-Quadruplex Sequences in Coding and Noncoding Regions Can Form G-Quadruplexes.

The HIV virus is one of the most studied viruses in the world. This is especially true in terms of gene sequencing, and to date more than 9 thousand genomic sequences of HIV isolates have been sequenced and analyzed. In this study, a series of DNA sequences, which have the potential to form G-quadruplex structures, is analyzed. Several such sequences were found in various coding and noncoding virus domains, including the U3 LTR, tat, rev, env, and vpx regions. Interestingly, a homological sequence to the already well-known HIV integrase aptamer was identified in the minus-strand. The sequences derived from original isolates were analyzed using standard spectral and electrophoretic methods. In addition, a recently developed methodology is applied which uses induced circular dichroism spectral profiles of G-quadruplex-ligand (Thiazole Orange) complexes to determine if G-rich sequences can adopt G-quadruplex structure. Targeting the G-quadruplexes or peptide domains corresponding to the G-rich coding sequence in HIV offers researchers attractive therapeutic targets which would be of particular use in the development of novel antiviral therapies. The analysis of G-rich regions can provide researchers with a path to find specific targets which could be of interest for specific types of virus.

G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA.

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5'-end also contributes essentially to the aptameric function.

Formation of highly ordered multimers in G-quadruplexes.

G-Rich DNA and RNA have a higher propensity to form G-quadruplex structures, but the presence of G-runs alone is not sufficient to prove that such sequences can form stable G-quadruplexes. While G-rich sequences are essential for G-quadruplex formation, not all G-rich sequences have the propensity to form G-quadruplex structures. In addition, monovalent metal ions, dehydrating agents, and loop sequences connecting the G-runs also play important roles in the topology of G-quadruplex folding. To date, no quantitative analysis of the CD spectra of G-quadruplexes in confrontation with the electrophoretic results has been performed. Therefore, in this study, we use information gained through the analysis of a series of well-known G-quadruplex-forming sequences to evaluate other less-studied sets of aptameric sequences. A simple and cost-effective methodology that can verify the formation of G-quadruplex motifs from oligomeric DNA sequences and a technique to determine the molecularity of these structures are also described. This methodology could be of great use in the prediction of G-quadruplex assembly, and the basic principles of our techniques can be extrapolated for any G-rich DNA sequences. This study also presents a model that can predict the multimerization of G-quadruplexes; the predictions offered by this model are shown to match the results obtained using circular dichroism.

Human papillomavirus G-quadruplexes.

Infection with human papillomaviruses (HPVs) is one of the most common sexually transmitted infections and can lead to development of head and neck, skin, and anogenital cancer, including cervical cancer, which represents one of the world's most significant health problems. In this study, we analyze G-rich regions in all known HPV genomes in order to evaluate their potential to fold into G-quadruplex structure. Interestingly, G-rich loci fulfilling the criteria for G-quadruplex formation were found in only 8 types of HPV. Nevertheless, viral G-quadruplexes in 7 sequences derived directly from HPVs are confirmed here for the first time. G-rich regions with the capacity to form G-quadruplexes are located in the LCR, L2, E1, and E4 regions of the HPV genome; therefore we assume that regulation processes in viruses could be affected by G-quadruplex formation. Our results represent a starting point for the design of specific ligands with viral G-quadruplex motifs and suggest novel methods for the control of viral replication and transcription.

Polyacrylamide temperature gradient gel electrophoresis.

Temperature Gradient Gel Electrophoresis (TGGE) is a form of electrophoresis in which temperature gradient is used to denature molecules as they move through either acrylamide or agarose gel. TGGE can be applied to analyze DNA, RNA, protein-DNA complexes, and, less commonly, proteins. Separation of double-stranded DNA molecules during TGGE relies on temperature-dependent melting of the DNA duplex into two single-stranded DNA molecules. Therefore, the mobility of DNA reflects not only the size of the molecule but also its nucleotide composition, thereby allowing separation of DNA molecules of similar size with different sequences. Depending on the relative orientation of electric field and temperature gradient, TGGE can be performed in either a parallel or a perpendicular mode. The former is used to analyze multiple samples in the same gel, whereas the later allows detailed analysis of a single sample. This chapter is focused on analysis of DNA by polyacrylamide TGGE using the perpendicular mode.

Novel naphthalimide polyamine derivatives as potential antitumor agents.

A novel series of naphthalimide polyamine conjugates were designed, synthesized and evaluated for in vitro antiproliferative activity against human leukemia (Jurkat), human cervical adenocarcinoma (HeLa), human breast adenocarcinoma (MCF-7) and human lung adenocarcinoma (A549) cell lines. From the six derivatives, the new I1 and A3 exhibited highest antiproliferative activity with the IC50 values of 5.67-11.02 µmol · L(-1). Cell cycle analysis of Jurkat cells exposed to I1 at a concentration of 30 µmol × L(-1) for 24 h exhibited a mild increase in S and G2/M fraction caused by accumulation of cells. This arrest was followed by an increase in sub-G0/G1 after 48 h of incubation. Jurkat cells exposed to A3 at a concentration of 30 µmol × L(-1) for 24 h showed an increase in G0/G1 fraction and after 48 h an increase in G2/M fraction followed by an increase in sub-G0/G1 after 72 h of incubation. Moreover, the A3 compound was observed to displace the intercalating agent ethidium bromide from calf thymus DNA using fluorescence spectroscopy. The apparent binding constant was estimated to be 3.1 × 10(6) M(-1) what indicates non-intercalating mode of DNA binding. On the other hand, we found no inhibitory effect of studied compounds on topoisomerase I and topoisomerase II activity. Finally, the localization of these compounds in the cells due to their inherent fluorescence was investigated with the fluorescence microscopy. Our results suggest that the naphthalimide polyamine conjugates rapidly penetrate to the cancer cells. Further studies are necessary to investigate the precise mechanism of action and to find out the relationship between the structure, character and position of substituents of naphthalimide polyamine conjugates and their biological activities.

Potential uses of G-quadruplex-forming aptamers.

Guanine quadruplex (G-quadruplex) structures are one of a number of structures which are capable of adopting aptamers. G-rich DNA or RNA has an increased propensity to form quadruplex structures which have unusual biophysical and biological properties. G-rich aptamers which form G-quadruplexes have several advantages over unstructured sequences: G-quadruplexes are non-immunogenic, thermodynamically and chemically stable and they have both higher resistance to various serum nucleases and an enhanced cellular uptake. These advantages have led to a number of synthetic oligonucleotides being studied for their potential use as therapeutic agents for cancer therapy and in the treatment of various other diseases. In addition to their suitability in the fields of medicine and biotechnology, these, highly specified, aptameric G-quadruplexes also have great potential in the further development of nano-devices; e.g. basic components in microarrays, microfluidics, sandwich assays and electrochemical biosensors. This review summarizes the biophysical properties of G-quadruplexes and highlights the importance of the stability and recognition properties of aptamers. Examples of the application of aptamers in medical therapy and in biosensors are also discussed.

Solution-state structure of an intramolecular G-quadruplex with propeller, diagonal and edgewise loops.

We herein report on the formation and high-resolution NMR solution-state structure determination of a G-quadruplex adopted by d[G(3)ATG(3)ACACAG(4)ACG(3)] comprised of four G-tracts with the third one consisting of four guanines that are intervened with non-G streches of different lengths. A single intramolecular antiparallel (3+1) G-quadruplex exhibits three stacked G-quartets connected with propeller, diagonal and edgewise loops of different lengths. The propeller and edgewise loops are well structured, whereas the longer diagonal loop is more flexible. To the best of our knowledge, this is the first high-resolution G-quadruplex structure where all of the three main loop types are present.

G-quadruplex motifs arranged in tandem occurring in telomeric repeats and the insulin-linked polymorphic region.

To date, various G-quadruplex structures have been reported in the human genome. There are numerous studies focusing on quadruplex-forming sequences in general, but few studies have focused on two or more quadruplexes in the same molecule, which are most commonly found in telomeric DNA and other tandem repeats, e.g., insulin-linked polymorphic region (ILPR). Although the human telomere consists of a number of repeats, higher-order G-quadruplex structures are discussed less often because of the complexity of the structures. In this study, sequences consisting of 4-12 repeats of d(G(4)TGT), d(G(3)T(2)A), and/or d(G(4)T(2)A) have been studied by circular dichroism, ultraviolet spectroscopy, and temperature-gradient gel electrophoresis. These sequences serve as a model for the arrangement of quadruplexes in the telomere and ILPR in solution. Our major findings are as follows. (i) The number of G-rich repeats has a great influence on G-quadruplex stability. (ii) The evidence of quadruplex-quadruplex interaction is confirmed. (iii) For the first time, we directly observed the melting behavior of different conformers in a single experiment. Our results agree with other calorimetric and spectroscopic data and data obtained by single-molecule studies, atomic force microscopy, and mechanical unfolding by optical tweezers. We propose that the end of telomeres can be formed by only a few tandem quadruplexes (fewer than three). Our findings improve our understanding of the mechanism of G-quadruplex formation in long repeats in G-rich-regulating parts of genes and telomere ends.

The circular dichroism and differential scanning calorimetry study of the properties of DNA aptamer dimers.

We have applied circular dichroism (CD), temperature-gradient gel electrophoresis (TGGE) and differential scanning calorimetry (DSC) to study the properties of novel bioengineered DNA aptamer dimers sensitive to fibrinogen (F) and heparin (H) binding sites of thrombin and compared them with canonical single stranded aptamer sensitive to fibrinogen binding site of thrombin (Fibri). The homodimer (FF) and heterodimer (FH) aptamers were constructed based on hybridization of their supported parts. CD results showed that both FF and FH dimers form stable guanine quadruplexes in the presence of potassium ions like those in Fibri. The thermal stability of aptamer dimers was slightly lower compared to those of canonical aptamers, but sufficient for practical applications. Both FF and FH aptamer dimers exhibited a potassium-dependent inhibitory effect on thrombin-mediated fibrin gel formation, which was on average two-fold higher than those of canonical single stranded Fibri aptamers.

The first derivative of a function of circular dichroism spectra: biophysical study of human telomeric G-quadruplex.

Depending on conditions and base modifications, telomeric repeats can form many topological structures; parallel, antiparallel and hybrid forms. The influence of salts and some specific ligands on conformational changes has already been established. In this study, we analyze the human telomeric repeats 5'-GGG(TTAGGG)(3)-3' because this sequence forms topologically different structures under various conditions which have been well described by many authors. CD results are compared with electrophoretic and UV absorption spectroscopy results obtained under corresponding conditions in the presence of different ratios of sodium and potassium ions and polyethylene glycol (PEG). We confirmed that the most stable G-quadruplexes could only form under crowding conditions with PEG-200 and K(+) ion, but the molecularity is increased. Other monovalent ions without the presence of K(+) are unable to form the parallel quadruplex conformer and no change of stoichiometry is observed, even when PEG-200 is present. The first derivative of a function applied to CD spectra seems to be a powerful tool for spectra evaluation of any G-quadruplex, and could be more unambiguous than a direct analysis of original spectra.

Evaluation of human telomeric g-quadruplexes: the influence of overhanging sequences on quadruplex stability and folding.

To date, various G-quadruplex structures have been reported in human telomeric sequences. Human telomeric repeats can form many topological structures depending on conditions and on base modification; parallel, antiparallel, and hybrid forms. The effect of salts and some specific ligands on conformational switches between different conformers is known, but the influence of protruding sequences has rarely been discussed. In this paper, we analyze different quadruplex-forming oligomers derived from human telomeric sequences which contain 3'- and 5'-protruding nucleotides, not usually associated with the G-quadruplex motif. The study was performed using electrophoresis, CD, and UV spectroscopies. The major findings are (i) protruding nucleotides destabilize the G-quadruplex structure, and (ii) overhanging sequences influence the folding of the quadruplex.

Structural features of intra- and intermolecular G-quadruplexes derived from telomeric repeats.

The 3' strand of telomeres is composed of tandem repeats of short G-rich sequences which protrude as single-stranded DNA overhangs. These repeats are G(3)T(2)A in humans and G(4)T(2) and G(4)T(4) in the ciliates Tetrahymena and Oxytricha, respectively. We analyzed different quadruplex-forming sequences derived from telomeric sequences, G(3+k)(T(n+k)G(3+k))(3) and G(3+k)(T(2)AG(3+k))(3), in the presence of Li(+), Na(+), and K(+) through the use of circular dichroism, UV spectroscopy, and electrophoresis, where k = 0 or 1 and n = 1-3. Results obtained under the given conditions can provide more detailed information about the quadruplex structure. The major findings are as follows. (i) G-Repeats in solution form a mix of topologically different structures; only G(3)(T(2)G(3))(3) and G(3)(TG(3))(3) repeats preferentially form the parallel interstrand structure. (ii) The Tetrahymena repeat can form at least two intramolecular conformers with different strand orientations and levels of stability. (iii) G-Quadruplex conformation and molecularity strongly depend on the type and concentration of ions used in the solution. The formation of intramolecular quadruplexes is governed by the length of the loops connecting G-runs. Intermolecular G-quadruplex forms are more likely to form in a higher concentration of ions for sequences where G-runs are separated by only one or two nucleotides.