Viktor Viglasky | Univerzita Pavla Jozefa Šafárika (original) (raw)

Papers by Viktor Viglasky

Biochimica et biophysica acta, 2017

Infection with high-risk human papillomaviruses (HPVs) can lead to development of cancer of the h... more 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. Combination of UV, CD and NMR spectroscopy and PAGE electrophoresis were used as they offer complementary insights into structural changes of G-rich oligonucleotides. 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...

Scientific Reports, 2016

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively,... more 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.

Journal of Biochemical and Biophysical Methods, 2005

J Biochem Biophys Meth, 2005

Temperature-gradient gel electrophoresis (TGGE) was used to study DNA-drug interactions. The resu... more Temperature-gradient gel electrophoresis (TGGE) was used to study DNA-drug interactions. The results indicate that at least two classes of DNA intercalating drugs are distinguishable with respect to temperature increase: reversible and irreversible. The method offers an excellent means of visualizing the melting profile of an individual DNA topoisomer in the presence of DNA binding drugs. Our findings coincide with UV/ VIS absorption spectroscopy data. D

FEBS Journal, 2008

G-rich regions appear in several locations in the human genome, including at the ends of linear c... more G-rich regions appear in several locations in the human genome, including at the ends of linear chromomes, the immunoglobin switch region, centromeres, fragile X syndrome repeats, and promoters of some genes . The sequences repeated in tandem, with three or four adjacent guanines, have been known to form polymorphic quadruplexes containing G-quartets stabilized by cyclic Hoogsteen hydrogen bondings. Quadruplex structures are highly stable DNA or RNA structures formed on G-rich sequences . The Na + and K + ions stabilize the stacking through their interactions with carbonyl oxygens of the eight guanines of two adjacent quartets . Direct evidence for the presence of G-quadruplex structures in vivo has been reported both at the telomeres of the ciliate Stylonychia [4] and those of humans , and at the promoter of c-myc . Moreover, other genomic regions were shown to be able to adopt quadruplex structures, such as the promoters of c-kit oncogene [6], HIF-1a [9], Bcl2 [10] and vascular endothelial growth factor .

Endocrine‚ Metabolic & Immune Disorders-Drug Targets, 2006

Despite the existence of a wealth of structural and theoretical data relating to palindromic sequ... more Despite the existence of a wealth of structural and theoretical data relating to palindromic sequences in the genome, the mechanism of cruciform formation in the presence of anthracyclines in miscellaneous biological processes is still poorly understood. Generally, DNA intercalators influence the DNA superhelicity, which plays a key role in the cruciform formation in DNA molecules. The potential of DNA intercalating ligands on the stabilization/destabilization of cruciform in DNA is discussed. Here, the indirect impact of anthracyclines to cell developing and surviving is analyzed for the first time. Primarily, the anthracycline modifies the helical properties of DNA and the overall DNA structure, and secondarily alters any cruciform-dependent processes, mainly DNA replication and transcription.

Journal of Virology, 2006

The Epstein-Barr virus (EBV) has been detected in subsets of breast cancers. In order to elaborat... more The Epstein-Barr virus (EBV) has been detected in subsets of breast cancers. In order to elaborate on these observations, we quantified by real-time PCR (Q-PCR) the EBV genome in biopsy specimens of breast cancer tissue as well as in tumor cells isolated by microdissection. Our findings show that EBV genomes can be detected by Q-PCR in about half of tumor specimens, usually in low copy numbers. However, we also found that the viral load is highly variable from tumor to tumor. Moreover, EBV genomes are heterogeneously distributed in morphologically identical tumor cells, with some clusters of isolated tumor cells containing relatively high genome numbers while other tumor cells isolated from the same specimen may be negative for EBV DNA. Using reverse transcription-PCR, we detected EBV gene transcripts: EBNA-1 in almost all of the EBV-positive tumors and RNA of the EBV oncoprotein LMP-1 in a smaller subset of the tissues analyzed. Moreover, BARF-1 RNA was detected in half of the cases studied. Furthermore, we observed that in vitro EBV infection of breast carcinoma cells confers resistance to paclitaxel (taxol) and provokes overexpression of a multidrug resistance gene (MDR1). Consequently, even if a small number of breast cancer cells are EBV infected, the impact of EBV infection on the efficiency of anticancer treatment might be of importance.

Journal of Biochemical and Biophysical Methods, 2005

Temperature-gradient gel electrophoresis (TGGE) was used to study DNA-drug interactions. The resu... more Temperature-gradient gel electrophoresis (TGGE) was used to study DNA-drug interactions. The results indicate that at least two classes of DNA intercalating drugs are distinguishable with respect to temperature increase: reversible and irreversible. The method offers an excellent means of visualizing the melting profile of an individual DNA topoisomer in the presence of DNA binding drugs. Our findings coincide with UV/ VIS absorption spectroscopy data. D

Biochemistry, 2013

Infection with human papillomaviruses (HPVs) is one of the most common sexually transmitted infec... more 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.

Analytical Biochemistry, 2007

Although there is a wealth of structural and theoretical data relating to palindromic sequences i... more Although there is a wealth of structural and theoretical data relating to palindromic sequences in genomes, the mechanisms of extrusion of cruciform structures during various biological processes in the presence of intercalating agents are still poorly understood. The current study addresses the effects of temperature and intercalator on cruciform extrusion from plasmids and also considers the effects of divalent metal ions on cruciform extrusion. It presents evidence that the cytotoxic effects of certain DNA binding drugs in vivo occur over concentration ranges corresponding to those that modulate cruciform extrusion in vitro. The results confirm earlier studies showing an inverse relationship between the effects of negative superhelicity and temperature on cruciform extrusion. By extrapolation, divalent metal ions facilitate cruciform extrusion by increasing superhelicity. The results allow the concentrations that preclude cruciform extrusion in DNA to be determined, and these are potentially informative about the relationships among temperature, DNA helical winding, cruciform formation, and intercalation. Overall, we provide new and interesting insights into the potential role of cruciform structures in biology and, by implication, cancer therapy.

Electrophoresis, 2002

Changes in DNA supercoiling might be essential to generate the response of cellular machinery to ... more Changes in DNA supercoiling might be essential to generate the response of cellular machinery to temperature stress. The heat-induced structural transition for a topoisomer depends on the value of its specific linking difference. We detect only less negatively supercoiled DNA and an abundance of alternative irregular DNA forms at culture temperatures close to the growth limit of Escherichia coli. We show that the irregular forms are derived from regular plasmid DNAs and their population in the cells is temperature-dependent. Here, we show that it is possible to isolate and characterize individual DNA topoisomers directly from cells without a topoisomerase treatment. Temperature gradient gel electrophoresis (TGGE) and atomic force microscopy (AFM) were used to study the effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability.

General physiology and biophysics, 2013

Guanine quadruplex (G-quadruplex) structures are one of a number of structures which are capable ... more 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.

Biochemistry, Jan 18, 2014

G-Rich DNA and RNA have a higher propensity to form G-quadruplex structures, but the presence of ... more 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...

Nucleic Acids Research, 2000

We have used temperature gradient gel electrophoresis (TGGE) to measure the progress of local den... more We have used temperature gradient gel electrophoresis (TGGE) to measure the progress of local denaturation in closed circular topoisomer DNA as a function of temperature and superhelicity (σ). We describe the versatility of this method as a tool for detecting various conformational modifications of plasmid DNAs. The early melting temperature of a structural transition for any topoisomer is dependent on the value of superhelicity. Supercoiled topoisomers represent a system of molecules that is sensitive to changes in temperature. We show that the topoisomer with the highest absolute value of superhelicity melts earlier than topoisomers with lower values. Thermal sensitivity of highly supercoiled plasmids could play a biologically important role in regulation of replication and expression in cells under thermal stress. The estimated melting temperature for plasmids with σ < -0.05 is very significant because these temperatures for early melting are below physiological temperatures.

Nucleic Acids Research, 2012

We herein report on the formation and high-resolution NMR solution-state structure determination ... more 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 highresolution G-quadruplex structure where all of the three main loop types are present.

Molecular Biology Reports, 2013

A novel series of naphthalimide polyamine conjugates were designed, synthesized and evaluated for... more 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.

European Biophysics Journal, 2011

Depending on conditions and base modifications, telomeric repeats can form many topological struc... more 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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-GGG(TTAGGG)(3)-3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; 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.

Electrophoresis, 2000

Heat-induced conformational transition of cytochrome c observed by temperature gradient gel elect... more Heat-induced conformational transition of cytochrome c observed by temperature gradient gel electrophoresis at acidic pH Temperature-gradient gel electrophoresis (TGGE) has been used to study the thermal unfolding of ferricytochrome c in low and high concentrations of acetic acid. It has been observed that the mobility of cytochrome c is a linear function of temperature when the system is characterized by a homogeneous population of conformation-state, single molecular species. Within the transition temperature range, the mobility clearly displays the characteristic sigmoidal shape describing the transitions of protein unfolding. The data obtained by TGGE were used to estimate the apparent thermodynamic parameters (enthalpy change DH vh and transition temperature T m ), associated with the transition of unfolding. The accuracy of the apparent thermodynamic parameters obtained by this method agrees within error limits with the values obtained by direct calorimetric measurements using differential scanning calorimetry (DSC).

ELECTROPHORESIS, 2003

The conformational stability of individual DNA topoisomers depends on the concentration of DNA in... more The conformational stability of individual DNA topoisomers depends on the concentration of DNA intercalating drugs. To study the DNA-drug interaction, we used ethidium bromide (EtBr) and negative supercoiled pUC19 as a model system. The effects of two anthracyclines widely used in cancer therapy, daunorubicin (Dau) and doxorubicin (Doxo), and EtBr were compared. In spite of their different chemical structures and intercalation mode, all intercalating agents show similar effects on the conformational stability of supercoiled DNA. Our observations show that the studied intercalators have at least two main effects on the supercoiled DNA: (i) they decrease the level of negative supercoiling and, at certain concentrations, they may induce positive supercoiling in DNA; (ii) a temperature increase can cause a recovery of negative supercoiling in DNA. The conformational stability of plasmid DNA-drug complexes has been investigated by temperature gradient gel electrophoresis (TGGE). We demonstrate the suitability of TGGE for this purpose, because it offers a global view on DNA-drug complexes over a continuous range of temperature. Images of DNA plasmids adsorbed onto a substrate at different temperatures and drug concentrations were acquired by atomic force microscopy (AFM), allowing us to distinguish directly the conformation chirality assumed by the plasmid under different conditions confirming TGGE results. Our detection system allows to characterize unknown drugs and to determine their intercalating properties.

Biophysical Chemistry, 2011

We have applied circular dichroism (CD), temperature-gradient gel electrophoresis (TGGE) and diff... more 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-* ACCEPTED MANUSCRIPT 2 mediated fibrin gel formation, which was on average two-fold higher than those of canonical single stranded Fibri aptamers.

Biochimica et biophysica acta, 2017

Infection with high-risk human papillomaviruses (HPVs) can lead to development of cancer of the h... more 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. Combination of UV, CD and NMR spectroscopy and PAGE electrophoresis were used as they offer complementary insights into structural changes of G-rich oligonucleotides. 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...

Scientific Reports, 2016

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively,... more 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&amp;amp;#39;-end also contributes essentially to the aptameric function.

Journal of Biochemical and Biophysical Methods, 2005

J Biochem Biophys Meth, 2005

Temperature-gradient gel electrophoresis (TGGE) was used to study DNA-drug interactions. The resu... more Temperature-gradient gel electrophoresis (TGGE) was used to study DNA-drug interactions. The results indicate that at least two classes of DNA intercalating drugs are distinguishable with respect to temperature increase: reversible and irreversible. The method offers an excellent means of visualizing the melting profile of an individual DNA topoisomer in the presence of DNA binding drugs. Our findings coincide with UV/ VIS absorption spectroscopy data. D

FEBS Journal, 2008

G-rich regions appear in several locations in the human genome, including at the ends of linear c... more G-rich regions appear in several locations in the human genome, including at the ends of linear chromomes, the immunoglobin switch region, centromeres, fragile X syndrome repeats, and promoters of some genes . The sequences repeated in tandem, with three or four adjacent guanines, have been known to form polymorphic quadruplexes containing G-quartets stabilized by cyclic Hoogsteen hydrogen bondings. Quadruplex structures are highly stable DNA or RNA structures formed on G-rich sequences . The Na + and K + ions stabilize the stacking through their interactions with carbonyl oxygens of the eight guanines of two adjacent quartets . Direct evidence for the presence of G-quadruplex structures in vivo has been reported both at the telomeres of the ciliate Stylonychia [4] and those of humans , and at the promoter of c-myc . Moreover, other genomic regions were shown to be able to adopt quadruplex structures, such as the promoters of c-kit oncogene [6], HIF-1a [9], Bcl2 [10] and vascular endothelial growth factor .

Endocrine‚ Metabolic & Immune Disorders-Drug Targets, 2006

Despite the existence of a wealth of structural and theoretical data relating to palindromic sequ... more Despite the existence of a wealth of structural and theoretical data relating to palindromic sequences in the genome, the mechanism of cruciform formation in the presence of anthracyclines in miscellaneous biological processes is still poorly understood. Generally, DNA intercalators influence the DNA superhelicity, which plays a key role in the cruciform formation in DNA molecules. The potential of DNA intercalating ligands on the stabilization/destabilization of cruciform in DNA is discussed. Here, the indirect impact of anthracyclines to cell developing and surviving is analyzed for the first time. Primarily, the anthracycline modifies the helical properties of DNA and the overall DNA structure, and secondarily alters any cruciform-dependent processes, mainly DNA replication and transcription.

Journal of Virology, 2006

The Epstein-Barr virus (EBV) has been detected in subsets of breast cancers. In order to elaborat... more The Epstein-Barr virus (EBV) has been detected in subsets of breast cancers. In order to elaborate on these observations, we quantified by real-time PCR (Q-PCR) the EBV genome in biopsy specimens of breast cancer tissue as well as in tumor cells isolated by microdissection. Our findings show that EBV genomes can be detected by Q-PCR in about half of tumor specimens, usually in low copy numbers. However, we also found that the viral load is highly variable from tumor to tumor. Moreover, EBV genomes are heterogeneously distributed in morphologically identical tumor cells, with some clusters of isolated tumor cells containing relatively high genome numbers while other tumor cells isolated from the same specimen may be negative for EBV DNA. Using reverse transcription-PCR, we detected EBV gene transcripts: EBNA-1 in almost all of the EBV-positive tumors and RNA of the EBV oncoprotein LMP-1 in a smaller subset of the tissues analyzed. Moreover, BARF-1 RNA was detected in half of the cases studied. Furthermore, we observed that in vitro EBV infection of breast carcinoma cells confers resistance to paclitaxel (taxol) and provokes overexpression of a multidrug resistance gene (MDR1). Consequently, even if a small number of breast cancer cells are EBV infected, the impact of EBV infection on the efficiency of anticancer treatment might be of importance.

Journal of Biochemical and Biophysical Methods, 2005

Temperature-gradient gel electrophoresis (TGGE) was used to study DNA-drug interactions. The resu... more Temperature-gradient gel electrophoresis (TGGE) was used to study DNA-drug interactions. The results indicate that at least two classes of DNA intercalating drugs are distinguishable with respect to temperature increase: reversible and irreversible. The method offers an excellent means of visualizing the melting profile of an individual DNA topoisomer in the presence of DNA binding drugs. Our findings coincide with UV/ VIS absorption spectroscopy data. D

Biochemistry, 2013

Infection with human papillomaviruses (HPVs) is one of the most common sexually transmitted infec... more 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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;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.

Analytical Biochemistry, 2007

Although there is a wealth of structural and theoretical data relating to palindromic sequences i... more Although there is a wealth of structural and theoretical data relating to palindromic sequences in genomes, the mechanisms of extrusion of cruciform structures during various biological processes in the presence of intercalating agents are still poorly understood. The current study addresses the effects of temperature and intercalator on cruciform extrusion from plasmids and also considers the effects of divalent metal ions on cruciform extrusion. It presents evidence that the cytotoxic effects of certain DNA binding drugs in vivo occur over concentration ranges corresponding to those that modulate cruciform extrusion in vitro. The results confirm earlier studies showing an inverse relationship between the effects of negative superhelicity and temperature on cruciform extrusion. By extrapolation, divalent metal ions facilitate cruciform extrusion by increasing superhelicity. The results allow the concentrations that preclude cruciform extrusion in DNA to be determined, and these are potentially informative about the relationships among temperature, DNA helical winding, cruciform formation, and intercalation. Overall, we provide new and interesting insights into the potential role of cruciform structures in biology and, by implication, cancer therapy.

Electrophoresis, 2002

Changes in DNA supercoiling might be essential to generate the response of cellular machinery to ... more Changes in DNA supercoiling might be essential to generate the response of cellular machinery to temperature stress. The heat-induced structural transition for a topoisomer depends on the value of its specific linking difference. We detect only less negatively supercoiled DNA and an abundance of alternative irregular DNA forms at culture temperatures close to the growth limit of Escherichia coli. We show that the irregular forms are derived from regular plasmid DNAs and their population in the cells is temperature-dependent. Here, we show that it is possible to isolate and characterize individual DNA topoisomers directly from cells without a topoisomerase treatment. Temperature gradient gel electrophoresis (TGGE) and atomic force microscopy (AFM) were used to study the effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability.

General physiology and biophysics, 2013

Guanine quadruplex (G-quadruplex) structures are one of a number of structures which are capable ... more 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.

Biochemistry, Jan 18, 2014

G-Rich DNA and RNA have a higher propensity to form G-quadruplex structures, but the presence of ... more 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...

Nucleic Acids Research, 2000

We have used temperature gradient gel electrophoresis (TGGE) to measure the progress of local den... more We have used temperature gradient gel electrophoresis (TGGE) to measure the progress of local denaturation in closed circular topoisomer DNA as a function of temperature and superhelicity (σ). We describe the versatility of this method as a tool for detecting various conformational modifications of plasmid DNAs. The early melting temperature of a structural transition for any topoisomer is dependent on the value of superhelicity. Supercoiled topoisomers represent a system of molecules that is sensitive to changes in temperature. We show that the topoisomer with the highest absolute value of superhelicity melts earlier than topoisomers with lower values. Thermal sensitivity of highly supercoiled plasmids could play a biologically important role in regulation of replication and expression in cells under thermal stress. The estimated melting temperature for plasmids with σ < -0.05 is very significant because these temperatures for early melting are below physiological temperatures.

Nucleic Acids Research, 2012

We herein report on the formation and high-resolution NMR solution-state structure determination ... more 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 highresolution G-quadruplex structure where all of the three main loop types are present.

Molecular Biology Reports, 2013

A novel series of naphthalimide polyamine conjugates were designed, synthesized and evaluated for... more 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.

European Biophysics Journal, 2011

Depending on conditions and base modifications, telomeric repeats can form many topological struc... more 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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-GGG(TTAGGG)(3)-3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; 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.

Electrophoresis, 2000

Heat-induced conformational transition of cytochrome c observed by temperature gradient gel elect... more Heat-induced conformational transition of cytochrome c observed by temperature gradient gel electrophoresis at acidic pH Temperature-gradient gel electrophoresis (TGGE) has been used to study the thermal unfolding of ferricytochrome c in low and high concentrations of acetic acid. It has been observed that the mobility of cytochrome c is a linear function of temperature when the system is characterized by a homogeneous population of conformation-state, single molecular species. Within the transition temperature range, the mobility clearly displays the characteristic sigmoidal shape describing the transitions of protein unfolding. The data obtained by TGGE were used to estimate the apparent thermodynamic parameters (enthalpy change DH vh and transition temperature T m ), associated with the transition of unfolding. The accuracy of the apparent thermodynamic parameters obtained by this method agrees within error limits with the values obtained by direct calorimetric measurements using differential scanning calorimetry (DSC).

ELECTROPHORESIS, 2003

The conformational stability of individual DNA topoisomers depends on the concentration of DNA in... more The conformational stability of individual DNA topoisomers depends on the concentration of DNA intercalating drugs. To study the DNA-drug interaction, we used ethidium bromide (EtBr) and negative supercoiled pUC19 as a model system. The effects of two anthracyclines widely used in cancer therapy, daunorubicin (Dau) and doxorubicin (Doxo), and EtBr were compared. In spite of their different chemical structures and intercalation mode, all intercalating agents show similar effects on the conformational stability of supercoiled DNA. Our observations show that the studied intercalators have at least two main effects on the supercoiled DNA: (i) they decrease the level of negative supercoiling and, at certain concentrations, they may induce positive supercoiling in DNA; (ii) a temperature increase can cause a recovery of negative supercoiling in DNA. The conformational stability of plasmid DNA-drug complexes has been investigated by temperature gradient gel electrophoresis (TGGE). We demonstrate the suitability of TGGE for this purpose, because it offers a global view on DNA-drug complexes over a continuous range of temperature. Images of DNA plasmids adsorbed onto a substrate at different temperatures and drug concentrations were acquired by atomic force microscopy (AFM), allowing us to distinguish directly the conformation chirality assumed by the plasmid under different conditions confirming TGGE results. Our detection system allows to characterize unknown drugs and to determine their intercalating properties.

Biophysical Chemistry, 2011

We have applied circular dichroism (CD), temperature-gradient gel electrophoresis (TGGE) and diff... more 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-* ACCEPTED MANUSCRIPT 2 mediated fibrin gel formation, which was on average two-fold higher than those of canonical single stranded Fibri aptamers.