Colorimetric Split G-Quadruplex Probes for Nucleic Acid Sensing: Improving Reconstituted DNAzyme’s Catalytic Efficiency via Probe Remodeling

Abstract

Split G-rich DNA probes can assemble into active peroxidase-mimicking DNAzymes in the presence of bioanalytes such as DNA, thereby providing a simple and cheap means to detect analytes in biological samples. A comprehensive study designed to reveal the salient probe architectural features and reaction conditions that facilitate facile reconstitution into enzymatically proficient enzymes unveiled these important findings: (a) The loops that connect the G3-tracts in a G-quadruplex structure can be replaced with a stem-loop or loop-stem-loop motif without destabilizing the resulting quadruplex structure; endowing the split G-rich probes with regions of limited complementarity leads to more proficient reconstituted enzymes. (b) The addition of hemin to antiparallel G-quadruplex DNAzymes lead to a blue shift in the CD spectra of the G-quadruplex DNAzymes. (c) The architectures of the DNA motifs that lie adjacent to the G-quadruplex structure influence both the stability and the enzymatic proficiency of the reconstituted enzymes. (d) The nature of the monovalent cation that is present in excess is a key determinant of the turnover number of the G-quadruplex DNAzyme; decomposition of G-quadruplex DNAzymes is slower in buffers that contain ammonium ions than those that contain sodium or potassium ions. These findings are important for the design of bioassays that use peroxidase-mimicking G-quadruplexes as detection labels.

DNA‐Based Peroxidation Catalyst—What Is the Exact Role of Topology on Catalysis and Is There a Special Binding Site for Catalysis?

“…Also, it remains to be shown if the peroxidation of organic molecules by G-quadruplexes takes place in a special binding pocket or site. Others have proposed that G-quadruplexes with anti-parallel topology are the active peroxidation catalysts [19,20] whereas our group [15,16] and others [12,14,21] have shown that G-quadruplexes with parallel or mixed-hybrid topologies are catalytically more proficient than anti-parallel G-quadruplexes. In this paper, we reveal that oligonucleotides that have a high propensity to form anti-parallel G-quadruplexes but are not peroxidase enzymes can indeed be turned into active peroxidation catalysts through the covalent attachment of hemin co-factor.…”

“…We [15] and others [23] have shown that oligonucleotide G1 [24] (5'-(G) 3 TA(G) 3 C(G) 3 TT(G) 3 T-3') mainly exists as a parallel or a mixed-hybrid conformer in the presence of monovalent cations (such as Na + , K + , or NH 4 + ). G1 binds to hemin and this is manifested spectroscopically as an increase in intensity as well as a slight red shift (398 to 404 nm) in the Soret band of hemin when increasing concentrations of G1 are added to hemin (see Figure 2 A).…”

“…[15] Therefore, we postulated that G-quadruplexes with parallel or mixed-hybrid structures, but not anti-parallel topology were the active peroxidation catalysts. [15] The Shangguan [12] and Kong [14] laboratories recently investigated over twenty Gquadruplex peroxidases with different topologies and have shown that in general, and in agreement with our earlier results, [15] the DNAzymes that formed parallel or mixedhybrid topologies were significantly better peroxidation catalysts than the ones that formed anti-parallel topologies. Taken together, the aforementioned studies suggest that the topology of G-quadruplexes is critical for their enzymatic activities.…”

“…[12][13][14][15][16] Thus far, the majority of G-quadruplex peroxidases reported to date are readily inactivated under the oxidizing reaction condition. [15,17,18] It is thought that hydroxyl radicals or the high-valent iron(IV)-oxo species generated during the peroxidation reaction are responsible for oxidizing the guanine tracts in G-quadruplexes to oxidized nucleobase analogues such as 8-oxo-guanines. 8-oxo-guanines do not support the G-quadruplex structure and hence peroxidations rates of G-quadruplex DNAzymes diminish as the guanines get oxidized during the course of the reaction.…”

“…We identify a new DNAzyme peroxidase (HT hemin1 ) (HT: human telomere repeat fragment), which is more resistant to oxidative inactivation compared to other G-quadruplexes. [15][16][17][18] These new findings challenge our current understanding of how nucleic acids catalyze cofactor oxidation reactions and also provide a starting point in the quest to Abstract: In the last decade, there has been growing interests in studies aimed at delineating the strategies used by various nucleic acid enzymes to facilitate catalysis. Insights gained from such studies would enable the design of better DNA/RNA catalysts for various applications such as biosensing.…”