Rimstone Laboratory

Federal Way, CT, United States

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Federal Way, CT, United States
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Kejnovska I.,Academy of Sciences of the Czech Republic | Vorlickova M.,Academy of Sciences of the Czech Republic | Vorlickova M.,Masaryk University | Brazdova M.,Academy of Sciences of the Czech Republic | Sagi J.,Rimstone Laboratory
Biopolymers | Year: 2014

For mimicking macromolecular crowding of DNA quadruplexes, various crowding agents have been used, typically PEG, with quadruplexes of micromolar strand concentrations. Thermal and thermodynamic stabilities of these quadruplexes increased with the concentration of the agents, the rise depended on the crowder used. A different phenomenon was observed, and is presented in this article, when the crowder was the quadruplex itself. With DNA strand concentrations ranging from 3 mM to 9 mM, the thermostability did not change up to 2 mM, above which it increased, indicating that the unfolding quadruplex units were not monomolecular above 2 mM. The results are explained by self-association of the G-quadruplexes above this concentration. The DGo 37 values, evaluated only below 2 mM, did not become more negative, as with the non-DNA crowders, instead, slightly increased. Folding topology changed from antiparallel to hybrid above 2 mM, and then to parallel quadruplexes at high, 6-9 mM strand concentrations. In this range, the concentration of the DNA phosphate anions approached the concentration of the K1 counterions used. Volume exclusion is assumed to promote the topological changes of quadruplexes toward the parallel, and the decreased screening of anions could affect their stability. ©2013 Wiley Periodicals, Inc.


Vorlickova M.,Academy of Sciences of the Czech Republic | Vorlickova M.,Masaryk University | Kejnovska I.,Academy of Sciences of the Czech Republic | Kejnovska I.,Masaryk University | And 7 more authors.
Methods | Year: 2012

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. © 2012 Elsevier Inc.


Babinsky M.,Masaryk University | Fiala R.,Masaryk University | Kejnovska I.,Masaryk University | Kejnovska I.,Academy of Sciences of the Czech Republic | And 7 more authors.
Nucleic Acids Research | Year: 2014

Abasic (AP) lesions are the most frequent type of damages occurring in cellular DNA. Here we describe the conformational effects of AP sites substituted for 2-deoxyadenosine in the first (ap7), second (ap13) or third (ap19) loop of the quadruplex formed in K+ by the human telomere DNA 5'-d[AG3(TTAG3)3]. CD spectra and electrophoresis reveal that the presence of AP sites does not hinder the formation of intramolecular quadruplexes. NMR spectra show that the structural heterogeneity is substantially reduced in ap7and ap19 as compared to that in the wild-type. These two (ap7 and ap19) sequences are shown to adopt the hybrid-1 and hybrid-2 quadruplex topology, respectively, with AP site located in a propeller-like loop. All three studied sequences transform easily into parallel quadruplex in dehydrating ethanol solution. Thus, the AP site in any loop region facilitates the formation of the propeller loop. Substitution of all adenines by AP sites stabilizes the parallel quadruplex even in the absence of ethanol. Whereas guanines are the major determinants of quadruplex stability, the presence or absence of loop adenines substantially influences quadruplex folding. The naturally occurring adenine-lacking sites in the human telomere DNA can change the quadruplex topology in vivo with potentially vital biological consequences. © The Author(s) 2014.


Konvalinova H.,Academy of Sciences of the Czech Republic | Konvalinova H.,Masaryk University | Dvorakova Z.,Academy of Sciences of the Czech Republic | Dvorakova Z.,Masaryk University | And 10 more authors.
Biochimie | Year: 2015

Abstract Various base lesions continuously form in cellular nucleic acids and the unrepaired lesions are promutagenic and procarcinogenic. Though natural base lesions have been extensively studied in double-stranded DNA models, these studies are only less than a decade old for non-canonical DNA models, such as quadruplexes. Here we present a report on the effects of three frequently occurring natural lesions that can form in the TTA loops on the structure of the human telomere quadruplex d[AG3(TTAG3)3]. We compared the effect of the abasic site and 8-oxoadenine replacing adenine and 5-hydroxymethyluracil substituting for thymine. The results showed that the three lesions impacted the stability and quadruplex folding in markedly different ways. The effects depended on the type of lesion and the position in the sequence. Analogous lesions of guanine in the G-tetrads extensively destabilized the quadruplex and the effects depended more on the position than on the type of lesion. The distinct effects of the loop substitutions as well as comparison of the modifications of the loops and the quadruplex tetrads are discussed in this communication. © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM).


Skolakova P.,Academy of Sciences of the Czech Republic | Bednarova K.,Academy of Sciences of the Czech Republic | Vorlickova M.,Academy of Sciences of the Czech Republic | Sagi J.,Rimstone Laboratory
Biochemical and Biophysical Research Communications | Year: 2010

This study was performed to evaluate how the loss of a guanine base affects the structure and stability of the three-tetrad G-quadruplex of 5'-dG3(TTAG3)3, the basic quadruplex-forming unit of the human telomere DNA. None of the 12 possible abasic sites hindered the formation of quadruplexes, but all reduced the thermodynamic stability of the parent quadruplex in both NaCl and KCl. The base loss did not change the Na+-stabilized intramolecular antiparallel architecture, based on CD spectra, but held up the conformational change induced in dG3(TTAG3)3 in physiological concentration of KCl. The reduced stability and the inhibited conformational transitions observed here in vitro for the first time may predict that unrepaired abasic sites in G-quadruplexes could lead to changes in the chromosome's terminal protection in vivo. © 2010 Elsevier Inc.


Sagi J.,Rimstone Laboratory | Renciuk D.,Academy of Sciences of the Czech Republic | Tomasko M.,Academy of Sciences of the Czech Republic | Vorlickova M.,Academy of Sciences of the Czech Republic
Biopolymers | Year: 2010

Replacement of two to four guanines by adenines in the human telomere DNA repeat dG 3(TTAG 3) 3 did not hinder the formation of quadruplexes if the substitutions took place in the terminal tetrad bridged by the diagonal loop of the intramolecular antiparallel three-tetrad scaffold, as proved by CD and PAGE in both Na + and K + solutions. Thermodynamic data showed that, in Na + solution, the dG 3(TTAG 3) 3 quadruplex was destabilized, the least by the two G:A:G:A tetrads, the most by the G:G:A:A tetrad in which the adenosines replaced synguanosines. In physiological K + solution, the highest destabilization was caused by the 4A tetrad. In K +, only the unmodified dG 3(TTAG 3) 3 quadruplex rearranged into a K +-dependent quadruplex form, none of the multiple adenine-modified structures did so. This may imply biological consequences for nonrepaired A-for-G mutations. © 2010 Wiley Periodicals, Inc.


Sagi J.,Rimstone Laboratory
Journal of Biomolecular Structure and Dynamics | Year: 2014

This review summarizes the results of structural studies carried out with analogs of G-quadruplexes built from natural nucleotides. Several dozens of base-, sugar-, and phosphate derivatives of the biological building blocks have been incorporated into more than 50 potentially quadruplex forming DNA and RNA oligonucleotides and the stability and folding topology of the resultant intramolecular, bimolecular and tetramolecular architectures characterized. The TG4T, TG5T, the 15 nucleotide-long thrombin binding aptamer, and the human telomere repeat AG3(TTAG3)3 sequences were modified in most cases, and four guanine analogs can be noted as being particularly useful in structural studies. These are the fluorescent 2-aminopurine, the 8-bromo-, and 8-methylguanines, and the hypoxanthine. The latter three analogs stabilize a given fold in a mixture of structures making possible accurate structural determinations by circular dichroism and nuclear magnetic resonance measurements. © 2013 Taylor & Francis.


Vorlickova M.,Academy of Sciences of the Czech Republic | Tomasko M.,Academy of Sciences of the Czech Republic | Sagi A.J.,Rimstone Laboratory | Bednarova K.,Academy of Sciences of the Czech Republic | Sagi J.,Rimstone Laboratory
FEBS Journal | Year: 2012

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′-dG 3(TTAG 3) 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 dG 3(TTAG 3) 3 by potassium ions, an observation that could imply biological relevance of the results. © 2011 FEBS.

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