Some G-quadruplex-hemin complexes can be used as peroxidase-mimicking DNAzymes, catalyzing H(2)O(2)-mediated reactions such as the oxidation of 2,2'-azinobis (3-ethylbenzothiozoline)-6-sulfonic acid (ABTS) by H(2)O(2). However, some challenges, for example, the relatively low catalytic activity and the disproportionation of the reaction product ABTS*(+), may seriously restrict further development and applications of these complexes. Here, we demonstrated the positive effect of adenosine triphosphate (ATP) on G-quadruplex-hemin DNAzyme-mediated catalytic reactions. The presence of ATP not only improved the catalytic activity of G-quadruplex-hemin DNAzymes, but also inhibited the disproportionation of ABTS*(+). These observations may improve the performance of existing G-quadruplex-hemin DNAzyme-based chemical sensors, for example, the Ag(+)-detection method that uses G-quadruplex-hemin DNAzymes, and widen the application range of G-quadruplex-hemin DNAzymes. We also demonstrated that the phosphate groups, nucleobase, and sugar of ATP determine the reaction-promoting ability of ATP. These observations may be helpful in the design of highly efficient enhancers for G-quadruplex-hemin DNAzymes.
Some G-quadruplex-hemin complexes are DNAzyme peroxidases that efficiently catalyze H(2)O(2)-mediated reactions, such as the oxidation of ABTS (2,2'-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid) by H(2)O(2). Since Ag(+) chelates guanine bases at the binding sites are involved in G-quadruplex formation, the presence of Ag(+) may disrupt these structures and inhibit the peroxidase activity of G-quadruplex-hemin DNAzymes. On the basis of this principle, a highly sensitive and selective Ag(+)-detection method was developed. The method allows simple detection of aqueous Ag(+) with a detection limit of 64 nM and a linear range of 50-3000 nM. Cysteine (Cys) is a strong Ag(+)-binder and competes with quadruplex-forming G-rich oligonucleotides for Ag(+)-binding, promoting the reformation of G-quadruplexes and increasing their peroxidase activity. Therefore, the Ag(+)-sensing system was also developed as a Cys-sensing system. This "turn-on" process allowed the detection of Cys at concentrations as low as 50 nM using a simple colorimetric technique. The Cys-sensing system could also be used for the detection of reduced glutathione (GSH). Neither the Ag(+)-sensing nor the Cys-sensing systems required labeled oligonucleotides. In addition, both gave large changes in absorbance signal that could be observed by the naked eye. Thus, a simple visual method for Ag(+)- or Cys-detection was developed.
G-rich sequences with the potential for quadruplex formation are common in genomic DNA. Considering that the biological functions of G-quadruplexes may well depend on their structures, the development of a sensitive structural probe for distinguishing different types of quadruplexes has received great attention. Crystal violet (CV) is a triphenylmethane dye, which can stack onto the two external G-quartets of a G-quadruplex. The ability of CV to discriminate G-quadruplexes from duplex and single-stranded DNAs has been reported by us. Herein, the ability of CV to discriminate parallel from antiparallel structures of a G-quadruplex was studied. The binding of CV to an antiparallel G-quadruplex can make its fluorescence intensity increase to a high level because of the protection of bound CV from the solvent by quadruplex end loops. The presence of side loops in parallel G-quadruplexes cannot provide bound CV such protection, causing the fluorescence intensity of CV/G-quadruplex mixture to be obviously weaker when the G-quadruplex adopts a parallel structure than that when the G-quadruplex adopts an antiparallel structure. Therefore, CV can be developed as a sensitive fluorescent biosensor for the discrimination of antiparallel G-quadruplexes from parallel G-quadruplexes and for monitoring the structural interconversion of G-quadruplexes. In addition, considering that some G-rich DNA sequences can adopt different G-quadruplex structures under Na(+) or K(+) ion conditions, a novel, cheap and simple K(+) ion detection method was developed. This method displays a high K(+) ion selectivity against Na(+) ion, the change of 200 mM in Na(+) ion concentration only causes a similar fluorescent signal change to 0.3 mM K(+) ion.
The structure-function relationship of G-quadruplex-hemin complexes with peroxidase activity was studied by comparing peroxidase activity and circular dichroism (CD) spectra of 22 oligonucleotides with the sequence of d(G(2)T(n))(3)G(2), d(G(3)T(n))(3)G(3) (n = 1-4) and dG(3)T(i)G(3)T(j)G(3)T(k)G(3). According to the experimental results, some conclusions can be drawn, such as the addition of hemin may promote the conversion of some G-quadruplexes from antiparallel structures to parallel structures; the formation of G-quadruplexes is a crucial factor in determining the peroxidase activity of G-quadruplex-hemin complexes; and the complexes formed by hemin and parallel G-quadruplexes have much higher peroxidase activity than those formed by hemin and antiparallel G-quadruplexes.
G-rich nucleic acid sequences with the potential to form G-quadruplex structures are common in biologically important regions. Most of these sequences are present with their complementary strands, so the development of a sensitive biosensor to distinguish G-quadruplex and duplex structures and to determine the competitive ability of quadruplex to duplex structures has received a great deal of attention. In this work, the interactions between two triphenylmethane dyes (malachite green (MG) and crystal violet (CV)) and G-quadruplex, duplex, or single-stranded DNAs were studied by fluorescence spectroscopy and energy-transfer fluorescence spectroscopy. Good discrimination between quadruplexes and duplex or single-stranded DNAs can be achieved by using the fluorescence spectrum of CV or the energy-transfer fluorescence spectra of CV and MG. In addition, by using energy-transfer fluorescence titrations of CV with G-quadruplexes, the binding-stoichiometry ratios of CV to G-quadruplexes can be determined. By using the fluorescence titrations of G-quadruplex-CV complexes with C-rich complementary strands, the fraction of G-rich oligonucleotide that engages in G-quadruplex structures in the presence of the complementary sequence can be measured. This study may provide a simple method for discrimination between quadruplexes and duplex or single-stranded DNAs and for measuring G-quadruplex percentages in the presence of the complementary C-rich sequences.
A potential bone tissue engineering material was produced from a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA), loaded with nanodiamond phospholipid compound (NDPC) via physical mixing. On the basis of hydrophobic effects and physical absorption, we modified the original hydrophilic surface of the nanodiamond (NDs) with phospholipids to be amphipathic, forming a typical core-shell structure. The ND-phospholipid weight ratio was optimized to generate sample NDPC50 (i.e., ND-phospholipid weight ratio of 100:50), and NDPC50 was able to be dispersed in a PLGA matrix at up to 20 wt %. Compared to a pure PLGA matrix, the introduction of 10 wt % of NDPC (i.e., sample NDPC50-PF10) resulted in a significant improvement in the material's mechanical and surface properties, including a decrease in the water contact angle from 80 to 55°, an approximately 100% increase in the Young's modulus, and an approximate 550% increase in hardness, thus closely resembling that of human cortical bone. As a novel matrix supporting human osteoblast (hFOB1.19) growth, NDPC50-PFs with different amounts of NDPC50 demonstrated no negative effects on cell proliferation and osteogenic differentiation. Furthermore, we focused on the behaviors of NDPC-PFs implanted into mice for 8 weeks and found that NDPC-PFs induced acceptable immune response and can reduce the rapid biodegradation of PLGA matrix. Our results represent the first in vivo research on ND (or NDPC) as nanofillers in a polymer matrix for bone tissue engineering. The high mechanical properties, good in vitro and in vivo biocompatibility, and increased mineralization capability suggest that biodegradable PLGA composite matrices loaded with NDPC may potentially be useful for a variety of biomedical applications, especially bone tissue engineering.
It has been reported that the complexes formed by hemin and some G‐quadruplexes can be developed as a new class of DNAzyme with peroxidase activity. This kind of DNAzyme has received a great deal of attention. But to date, the actual G‐quadruplex structure that can provide hemin with enhanced peroxidase activity is in doubt. Herein, the G‐quadruplex structure of CatG4, a 21‐nucleotide DNA oligomer which was previously reported to bind hemin and the resulting complex exhibiting enhanced peroxidase activity, was characterized by fluorescence and circular dichroism measurements. The results suggest that the catalytically active form of CatG4 may be a unimolecular parallel quadruplex rather than a unimolecular chair‐type antiparallel quadruplex or a multistranded parallel quadruplex. In addition, the fluorescence analysis of labeled oligonucleotides may be developed as a supplementary tool for the study of DNA conformations. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 331–339, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
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