Deciphering the DNAzyme Activity of Multimeric Quadruplexes: Insights into Their Actual Role in the Telomerase Activity Evaluation Assay

Abstract

The end of human telomeres is comprised of a long G-rich single-stranded DNA (known as 3'-overhang) able to adopt an unusual three-dimensional "beads-on-the-string" organization made of consecutively stacked G-quadruplex units (so-called quadruplex multimers). It has been widely demonstrated that, upon interaction with hemin, discrete quadruplexes acquire peroxidase-mimicking properties, oxidizing several organic probes in H(2)O(2)-rich conditions; this property, known as DNAzyme, has found tens of applications in the last two decades. However, little is known about the DNAzyme activity of multimeric quadruplexes; this is an important question to address, especially in light of recent reports that exploit the DNAzyme process to optically assess the activity of an enzyme that elongates the telomeric overhang, the telomerase. Herein, we thoroughly investigate the DNAzyme activity of long telomeric fragments, with a particular focus on both the nature of the hemin/multimeric quadruplex interactions and the putative higher-order fold of the studied fragments; in light of our results, we also propose possible ways that may be followed to improve the use of DNAzyme to evaluate the telomerase activity.

Insight into G-quadruplex-hemin DNAzyme/RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity

“…The nearly identical titration curves of Soret absorption band of hemin in response to Dz-00 and Dz-11 imply that these sequences bind hemin with similar affinity (Supplementary Figure S4). Further Job plot (Supplementary Figure S5A) and Scatchard analysis (Supplementary Figure S5B and C) indicate that the binding stoichiometry of hemin/DNA is 1:1 for both Dz-00 and Dz-11, consistent with the stoichiometry found in other G4 DNAzymes (2,26,35) and the calculated dissociation constant ( K d ) of Dz-00-hemin (36 ± 2 nM) is almost the same as that of Dz-11-hemin (30 ± 3 nM). The unchanged hemin-binding affinity, in combination with the electronic absorption spectra of Dz-11 and Dz-00, indicates that the additional 3′A does not directly participate in the binding of hemin.…”

“…As a result, a number of G4 sequences with potent activity have been discovered and the topological analysis on these G4 sequences indicates that parallel G4 structure is preferable for high peroxidase activity. Another popular strategy is the addition of exogenous activity-boosting agents, such as nitrogenous buffers (2), adenosine triphosphate (24,25), DOTASQ (26,27) and spermine (28). This strategy is intriguingly useful, but its implementation usually requires high concentration of the boosting agents because of the intermolecular enhancing effect.…”

An electrochemical aptasensor for thrombin using synergetic catalysis of enzyme and porous Au@Pd core–shell nanostructures for signal amplification

“…Owing to the large surface area, porous Au@Pd core-shell nanostructures were firstly used as the nanocarrier for the immobilization of amino terminated TBA (NH 2 -TBA) and electroactive toluidine blue (Tb), followed by the intercalation of hemin to form hemin/G-quadruplex with favorable peroxidase-mimicking properties (Stefan et al, 2011). Subsequently, GOx acted as the blocking reagent to block the remaining active sites on the porous Au@Pd core-shell nanostructures, achieving the secondary TB aptamer (Tb, GOx and hemin/ G-quadruplex multi-labeled Au@Pd bioconjugates).…”

Dinickel–Salphen Complexes as Binders of Human Telomeric Dimeric G‐Quadruplexes

“…Furthermore, it should also be noted that complexes 2 c and 1 exhibited higher than or comparable affinities/thermal stabilization than other G2T1 binders previously reported in the literature (Table S2 in the Supporting Information) 24, 26, 27, 29, 30, 31…”

“…Though a large number of small molecules have been previously developed as single G‐quadruplex DNA binders,5, 6, 7, 8, 9 comparatively very few have been studied (or indeed specifically designed) as binders for multimeric G‐quadruplex structures 24, 25, 26, 27, 28, 29, 30, 31, 32. This includes a chiral cyclic helicene proposed to bind in the cleft between two human telomeric G‐quadruplexes linked by a TTA spacer 25.…”