36 research outputs found
Hydration Changes upon DNA Folding Studied by Osmotic Stress Experiments
AbstractThe thermal stability of nucleic acid structures is perturbed under the conditions that mimic the intracellular environment, typically rich in inert components and under osmotic stress. We now describe the thermodynamic stability of DNA oligonucleotide structures in the presence of high background concentrations of neutral cosolutes. Small cosolutes destabilize the basepair structures, and the DNA structures consisting of the same nearest-neighbor composition show similar thermodynamic parameters in the presence of various types of cosolutes. The osmotic stress experiments reveal that water binding to flexible loops, unstable mismatches, and an abasic site upon DNA folding are almost negligible, whereas the binding to stable mismatch pairs is significant. The studies using the basepair-mimic nucleosides and the peptide nucleic acid suggest that the sugar-phosphate backbone and the integrity of the basepair conformation make important contributions to the binding of water molecules to the DNA bases and helical grooves. The study of the DNA hydration provides the basis for understanding and predicting nucleic acid structures in nonaqueous solvent systems
Mirror-image dependence : targeting enantiomeric G-quadruplex DNA using triplex metallohelices
Natural dâDNA and lâDNA are mirrorâimage counterparts. However, because of the inherent flexibility and conformation diversity of DNA, it is still not clear how enantiomeric compounds recognize dâDNA and lâDNA. Herein, taking Gâquadruplex (G4) DNA as an example that has diverse conformations and distinct biofunctions, the binding of ten pairs of iron triplex metallohelices to dâ and lâG4 DNA were evaluated. The Îâenantiomer binds to dâDNA and the Îâenantiomer binds to lâDNA, exhibiting almost the same stabilization effect and binding affinity. The binding affinity of the Îâmetallohelix with dâG4 is nearly 70âfold higher than that of Îâmetallohelix binding dâG4. ÎâMetallohelix binding to dâG4 follows a twoâstep binding process driven by a favorable enthalpy contribution to compensate for the associated unfavorable entropy
Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers
Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed
Newly characterized interaction stabilizes DNA structure: oligoethylene glycols stabilize G-quadruplexes CHâÏ interactions
Oligoethylene glycols are used as crowding agents in experiments that aim to understand the effects of intracellular environments on DNAs. Moreover, DNAs with covalently attached oligoethylene glycols are used as cargo carriers for drug delivery systems. To investigate how oligoethylene glycols interact with DNAs, we incorporated deoxythymidine modified with oligoethylene glycols of different lengths, such as tetraethylene glycol (TEG), into DNAs that form antiparallel G-quadruplex or hairpin structures such that the modified residues were incorporated into loop regions. Thermodynamic analysis showed that because of enthalpic differences, the modified G-quadruplexes were stable and the hairpin structures were slightly unstable relative to unmodified DNA. The stability of G-quadruplexes increased with increasing length of the ethylene oxides and the number of deoxythymidines modified with ethylene glycols in the G-quadruplex. Nuclear magnetic resonance analyses and molecular dynamics calculations suggest that TEG interacts with bases in the G-quartet and loop via CH-pi and lone pair-pi interactions, although it was previously assumed that oligoethylene glycols do not directly interact with DNAs. The results suggest that numerous cellular co-solutes likely affect DNA function through these CH-pi and lone pair-pi interactions
Choline Dihydrogen Phosphate Destabilizes GâQuadruplexes and Enhances Transcription Efficiency <i>In Vitro</i> and in Cells
G-quadruplexes in disease-related genes are associated
with various
biological processes and regulate disease progression. Although methods
involving ligands and other techniques are available to stabilize
G-quadruplexes, approaches for destabilizing G-quadruplexes remain
limited. Here, we evaluated whether G-quadruplexes can be destabilized
using choline dihydrogen phosphate (choline dhp), a highly biocompatible
hydrated ionic liquid. Circular dichroism spectral measurements at
increasing temperatures revealed that choline dhp destabilized G-quadruplexes
more effectively than did KCl-containing solutions. Thermodynamic
analysis indicated that destabilization occurred via an entropic contribution,
suggesting that choline ions did not coordinate with the G-quartets,
because of their large radii. Subsequently, plasmid DNAs containing
G-quadruplexes were constructed, and transcription reactions were
performed in nuclear extracts from living cells. G-quadruplexes repressed
transcription, whereas the addition of choline dhp increased transcription.
Although ionic liquids often inactivate biomolecules, choline dhp
can be used to culture various cells. Furthermore, the transcription
of template DNA containing the G-quadruplex was greatly enhanced in
living MDA-MD-231 cells (aggressive human breast cancer cells) cultured
with choline dhp. Our results show that choline dhp destabilizes G-quadruplexes
in cells, indicating that choline dhp can regulate gene expression.
Thus, choline dhp may be useful for regulating target disease-related
genes
C-Rich Sequence in a Non-Template DNA Strand Regulates Structure Change of G-Quadruplex in a Template Strand during Transcription
New Modified Deoxythymine with Dibranched Tetraethylene Glycol Stabilizes G-Quadruplex Structures
Methods for stabilizing G-quadruplex formation is a promising therapeutic approach for cancer treatment and other biomedical applications because stable G-quadruplexes efficiently inhibit biological reactions. Oligo and polyethylene glycols are promising biocompatible compounds, and we have shown that linear oligoethylene glycols can stabilize G-quadruplexes. Here, we developed a new modified deoxythymine with dibranched or tribranched tetraethylene glycol (TEG) and incorporated these TEG-modified deoxythymines into a loop region that forms an antiparallel G-quadruplex. We analyzed the stability of the modified G-quadruplexes, and the results showed that the tribranched TEG destabilized G-quadruplexes through entropic contributions, likely through steric hindrance. Interestingly, the dibranched TEG modification increased G-quadruplex stability relative to the unmodified DNA structures due to favorable enthalpic contributions. Molecular dynamics calculations suggested that dibranched TEG interacts with the G-quadruplex through hydrogen bonding and CH-pi interactions. Moreover, these branched TEG-modified deoxythymine protected the DNA oligonucleotides from degradation by various nucleases in human serum. By taking advantage of the unique interactions between DNA and branched TEG, advanced DNA materials can be developed that affect the regulation of DNA structure
A Turn-On Detection of DNA Sequences by Means of Fluorescence of DNA-Templated Silver Nanoclusters via Unique Interactions of a Hydrated Ionic Liquid
Nucleic acid stability and structure, which are crucial to the properties of fluorescent DNA-templated silver nanoclusters (DNA-Ag NCs), significantly change in ionic liquids. In this work, our purpose was to study DNA-Ag NCs in a buffer containing the hydrated ionic liquid of choline dihydrogen phosphate (choline dhp) to improve fluorescence for application in DNA detection. Due to the stabilisation of an i-motif structure by the choline cation, a unique fluorescence emission—that was not seen in an aqueous buffer—was observed in choline dhp and remained stable for more than 30 days. A DNA-Ag NCs probe was designed to have greater fluorescence intensity in choline dhp in the presence of a target DNA. A turn-on sensing platform in choline dhp was built for the detection of the BRCA1 gene, which is related to familial breast and ovarian cancers. This platform showed better sensitivity and selectivity in distinguishing a target sequence from a mutant sequence in choline dhp than in the aqueous buffer. Our study provides new evidence regarding the effects of structure on properties of fluorescent DNA-Ag NCs and expands the applications of fluorescent DNA-Ag NCs in an ionic liquid because of improved sensitivity and selectivity
Effect of the G-quadruplex stability on the production of slipped and arrested transcripts.
<p>Denaturing gel electrophoresis of products of transcription reactions carried out for 90°C using Q5 template. Reaction mixtures contained 0.3 ”M T7 polymerase and 1.5 ”M DNA template in a buffer containing 40 mM Tris-HCl (pH 8.0), 8 mM MgCl<sub>2</sub>, and 2 mM spermidine and various concentrations of (a) KCl or (b) LiCl. Lane 1 shows 10-nt size marker, lane 2 shows 35-nt RNA, and lanes 3 to 7 show transcription products in the presence of 0, 10, 30, 50, and 70 mM (a) KCl or (b) LiCl. Blue and red arrows indicate the slipped and arrested transcripts, respectively.</p