Cationic Porphyrin-Mediated G-Quadruplex DNA Oxidative Damage: Regulated by the Initial Interplay between DNA and TMPyP4.

G-quadruplex (G4) ligand-induced DNA damage has been involved in many physiological functions of cells. Herein, cationic porphyrin (TMPyP4)-mediated DNA oxidation damage was investigated aiming at mitochondrial G4 DNA (mt9438) and its structural analogue of the thrombin-binding aptamer (TBA). TMPyP4 is found to stabilize TBA G4 but destabilize mt9438. For two resulting DNA-TMPyP4 assemblies, the distinct light-induced singlet oxygen (1O2) generation and the subsequent DNA damage were found. For mt9438-TMPyP4, a slower 1O2-induced DNA damage takes place and results in the formation of DNA aggregation. In contrast, 1O2 tends to promote DNA unfolding in a relatively faster rate for TBA-TMPyP4. Despite of such distinct DNA damage behavior, UV resonance Raman spectra reveal that for both mt9438-TMPyP4 and TBA-TMPyP4 the DNA damage commonly stems from the guanine-specific oxidation. Our results clearly indicate that the ligand-mediated DNA damage is strongly dependent on the initial interplay between DNA and the ligand.

Controllable stereoinversion in DNA-catalyzed olefin cyclopropanation via cofactor modification.

The assembly of DNA with metal-complex cofactors can form promising biocatalysts for asymmetric reactions, although catalytic performance is typically limited by low enantioselectivities and stereo-control remains a challenge. Here, we engineer G-quadruplex-based DNA biocatalysts for an asymmetric cyclopropanation reaction, achieving enantiomeric excess (eetrans) values of up to +91% with controllable stereoinversion, where the enantioselectivity switches to -72% eetrans through modification of the Fe-porphyrin cofactor. Complementary circular dichroism, nuclear magnetic resonance, and fluorescence titration experiments show that the porphyrin ligand of the cofactor participates in the regulation of the catalytic enantioselectivity via a synergetic effect with DNA residues at the active site. These findings underline the important role of cofactor modification in DNA catalysis and thus pave the way for the rational engineering of DNA-based biocatalysts.

Highly Selective Detection of K+ Based on a Dimerized G-Quadruplex DNAzyme.

Potassium ion (K+) plays a crucial role in biological systems, such as maintaining cellular processes and causing diseases. However, specifically, the detection of K+ is extremely challenging because of the coexistence of the chemically similar ion of Na+ under physiological conditions. In this work, a K+ specific biosensor is constructed on the basis of a dimerized G-quadruplex (GQ) DNA, which is promoted by K+, and the enzymatic activity of the resulting DNAzyme depends on the concentration of the K+. The K+ in a 1-200 mM concentration range can be selectively detected by visual color, UV-Vis absorbance or fluorescence even if the concentration of the accompanying Na+ is up to 140 mM at an ambient condition up to 45 °C. In addition, this system can also be used to selectively detect NH4+ in a 5-200 mM concentration range. This dimerized DNAzyme offers a new type of biosensor with a potential application in the biological system.

The noncovalent dimerization of a G-quadruplex/hemin DNAzyme improves its biocatalytic properties.

While many protein enzymes exert their functions through multimerization, which improves both selectivity and activity, this has not yet been demonstrated for other naturally occurring catalysts. Here, we report a multimerization effect applied to catalytic DNAs (or DNAzymes) and demonstrate that the enzymatic efficiency of G-quadruplexes (GQs) in interaction with the hemin cofactor is remarkably enhanced by homodimerization. The resulting non-covalent dimeric GQ-DNAzyme system provides hemin with a structurally defined active site in which both the cofactor (hemin) and the oxidant (H2O2) are activated. This new biocatalytic system efficiently performs peroxidase- and peroxygenase-type biotransformations of a broad range of substrates, thus providing new perspectives for biotechnological application of GQs.

Loop permutation affects the topology and stability of G-quadruplexes.

G-quadruplexes are unusual DNA and RNA secondary structures ubiquitous in a variety of organisms including vertebrates, plants, viruses and bacteria. The folding topology and stability of intramolecular G-quadruplexes are determined to a large extent by their loops. Loop permutation is defined as swapping two or three of these regions so that intramolecular G-quadruplexes only differ in the sequential order of their loops. Over the past two decades, both length and base composition of loops have been studied extensively, but a systematic study on the effect of loop permutation has been missing. In the present work, 99 sequences from 21 groups with different loop permutations were tested. To our surprise, both conformation and thermal stability are greatly dependent on loop permutation. Loop permutation actually matters as much as loop length and base composition on G-quadruplex folding, with effects on Tm as high as 17°C. Sequences containing a longer central loop have a high propensity to adopt a stable non-parallel topology. Conversely, sequences containing a short central loop tend to form a parallel topology of lower stability. In addition, over half of interrogated sequences were found in the genomes of diverse organisms, implicating their potential regulatory roles in the genome or as therapeutic targets. This study illustrates the structural roles of loops in G-quadruplex folding and should help to establish rules to predict the folding pattern and stability of G-quadruplexes.

Fluorescence Spectroscopic Insight into the Supramolecular Interactions in DNA-Based Enantioselective Sulfoxidation.

Interactions of copper(II)-bipyridine cofactors and thioanisole substrate with human telomeric G-quadruplex DNA were studied by UV/Vis absorption, circular dichroism, and fluorescence quenching titration. Three copper(II)-bipyridine complexes are equivalently anchored to the G-quadruplex scaffold at all five fluorescently labeled sites. Thioanisole interacts with the DNA architecture at both the second loop and 3' terminus in the absence or presence of copper(II)-bipyridine complexes. These nonspecificities in the weak interactions of CuII complexes and thioanisole with G-quadruplex might explain why DNA only affords a modest enantioselectivity in the oxidation of thioanisole. These findings provide insights toward the construction of highly enantioselective DNA-based catalysts.

Probing the interaction of copper cofactor and azachalcone substrate with G-quadruplex of DNA based Diels-Alderase by site-specific fluorescence quenching titration.

DNAzymes have been widely used in biosensors, asymmetric synthesis and pharmaceuticals. Typically, metal cofactor and substrate interact with DNA by supramolecular interactions in DNAzyme based asymmetric catalysis. However, binding positions of cofactor and substrate with DNA scaffold are not well understood, which is an obstacle to reveal the assembly and catalysis mechanisms of DNAzyme. Herein, we report a method of site-specific fluorescence quenching titration to elucidate the assembly and catalysis processes of a G-quadruplex based Diels-Alderase DNAzyme. Titration data indicate that cofactor Cu(II)-terpyridine stacked atop 5' and 3' external G-quartets with high and low binding affinities respectively, and induced the G-quadruplex to form a hybrid-1 topology. Substrate azachalcone interacted with 3' quartet exclusively, implicating that asymmetric Diels-Alder cycloaddition may occur at 3' G-quartet. In addition, enzyme kinetic analyses show that activity and enantioselectivity of the DNAzyme were substantially preserved after attaching the fluorophores. Overall, site-specific fluorescence quenching is a concise and efficient approach to probe the assembly processes of DNAzyme.

Higher-order human telomeric G-quadruplex DNA metalloenzyme catalyzed Diels-Alder reaction: an unexpected inversion of enantioselectivity modulated by K(+) and NH4(+) ions.

We report that K(+) and NH4(+) present different allosteric activation for higher-order human telomeric G-quadruplex DNA metalloenzyme. The obtained major endo products of Diels-Alder reaction can be switched from one preferred configuration in K(+) media (up to 92% ee) to its mirror configuration in NH4(+) media (up to -90% ee).

Terpyridine-Cu(ii) targeting human telomeric DNA to produce highly stereospecific G-quadruplex DNA metalloenzyme.

The cofactors commonly involved in natural enzymes have provided the inspiration for numerous advances in the creation of artificial metalloenzymes. Nevertheless, to design an appropriate cofactor for a given biomolecular scaffold or vice versa remains a challenge in developing efficient catalysts in biochemistry. Herein, we extend the idea of G-quadruplex-targeting anticancer drug design to construct a G-quadruplex DNA metalloenzyme. We found that a series of terpyridine-Cu(ii) complexes (CuLn) can serve as excellent cofactors to dock with human telemetric G-quadruplex DNA. The resulting G-quadruplex DNA metalloenzyme utilising CuL1 catalyzes an enantioselective Diels-Alder reaction with enantioselectivity of >99% enantiomeric excess and about 73-fold rate acceleration compared to CuL1 alone. The terpyridine-Cu(ii) complex cofactors demonstrate dual functions, both as an active site to perform catalysis and as a structural regulator to promote the folding of human telemetric G-quadruplex DNA towards excellent catalysts.