Sylvia Omonirume Malomo

Split G-quadruplex DNAzymes offer unique opportunities for building gated biosensors with a wide range of applications. Splitting G4 DNAzymes involves separating guanine tracts in the G-quadruplex DNA sequence into two non-functional sequences that reconstitute into a functional G-quadruplex with peroxidase activity upon hybridisation of the aptamer probe region within the split system with the target molecule. Several studies have demonstrated the reassembly of split G4 DNAzymes and their applications in the detection of various analytes. This approach offers unique opportunities for modular biosensor construction, target-dependent activation, lack of requirement for labelling, amplification-free high sensitivity, and specificity over traditional G4 sensing. In this review, we explore the strategies of splitting G-quadruplex and their applications in biomedical diagnosis, environmental sensing, food safety monitoring, cell detection, and the integration of the technology with nanomaterials for enhanced stability and sensitivity. We considered the classical intermolecular split strategies that utilise binary probes and intramolecular split systems, which integrate the spacer DNA that allow for single probes as the model G4 sequence. Finally, we explore the current challenges required to develop split G-quadruplex DNAzymes into tools for routine practical applications.

G-quadruplex (G4) DNAzymes, guanine-rich sequences that fold into four-stranded structures and bind hemin to mimic peroxidase activity, are widely used in biosensing. Split G4 DNAzymes offer conditional activation upon target recognition, enabling high specificity and modularity. However, achieving low OFF-state leakage remains a major challenge. Here, we systematically characterized four representative G4 scaffolds, C-myc, Bcl2, PS5.M, and C-kit, under standardized ABTS/H₂O₂ conditions to assess their kinetic properties and suitability for split designs. C-myc exhibited the highest sustained activity and near-linear concentration dependence, making it ideal for quantitative sensing, while Bcl2 showed durable catalysis suited for extended read windows. C-kit produced rapid bursts with early plateaus, favoring binary outputs, and PS5.M initiated quickly but inactivated rapidly, suggesting potential application of systems requiring fast response. Split-mode analysis revealed that symmetric 2:2 partitions often retained significant activity, whereas asymmetric 3:1 splits reduced but did not eliminate leakage. Among the four G4 DNAzymes, PS5.M demonstrated the most promising OFF-state suppression. Design strategies to minimize leakage including non-classical splits, loop/flank edits, and template-assisted assembly could be used to optimize biosensor functionalities. These findings identify essential factors critical for designing robust split DNAzyme biosensors, advancing applications in diagnostics and molecular logic gates.

G-quadruplex (G4) DNAzymes with peroxidase activities hold potential for applications in biosensing. While these nanozymes are easy to assemble, they are not as efficient as natural peroxidase enzymes. Several approaches are being used to better understand the structural basis of their reaction mechanisms, with a view to designing constructs with improved catalytic activities. Spermine alters the structures and enhances the activities of some G4 DNAzymes. The reported effect of spermine in shifting the conformation of some G4 DNAzymes from antiparallel to parallel has not been tested on multimeric G4 DNAzymes. In this study, we examined the effects of spermine on the catalytic activities of multivalent constructs of Bcl2, c-MYC, PS2.M, and PS5.M. Our findings show that spermine significantly improved the peroxidase activity of PS2.M, an antiparallel G4 DNAzyme, while there was no significant effect on c-MYC, which already exists in a parallel conformation. The addition of spermine led to a substantial increase in the initial velocity of PS2.M and its multimeric form, enhancing it by approximately twofold. Therefore, spermine enhancement offers promise in expanding the range of DNAzymes available for use as biosensing tools.

To search for a novel thermostable esterase for optimized industrial applications, esterase from a thermophilic eubacterium species, Thermoanaerobacter tengcongensis MB4, was purified and characterized in this work. Sequence analysis of T. tengcongensis esterase with other homologous esterases of the same family revealed an apparent tail at the C-terminal that is not conserved across the esterase family. Hence, it was hypothesized that the tail is unlikely to have an essential structural or catalytic role. However, there is no documented report of any role for this tail region. We probed the role of the C-terminal domain on the catalytic activity and substrate preference of T. tengcongensis esterase EstA3 with a view to see how it could be engineered for enhanced properties. To achieve this, we cloned, expressed, and purified the wild-type and the truncated versions of the enzyme. In addition, a naturally occurring member of the family (from Brevibacillus brevis) that lacks the C-terminal tail was also made. In vitro characterization of the purified enzymes showed that the C-terminal domain contributes significantly to the catalytic activity and distinct substrate preference of T. tengcongensis esterase EstA3. All three recombinant enzymes showed the highest preference for paranitrophenyl butyrate (pNPC4), which suggests they are true esterases, not lipases. Kinetic data revealed that truncation had a slight effect on the substrate-binding affinity. Thus, the drop in preference towards long-chain substrates might not be a result of substrate binding affinity alone. The findings from this work could form the basis for future protein engineering allowing the modification of esterase catalytic properties through domain swapping or by attaching a modular protein domain.

Protein tyrosine phosphatases (PTPs) of the polymerase and histidinol phosphatase (PHP) superfamily with characteristic phosphatase activity dependent on divalent metal ions are found in many Gram-positive bacteria. Although members of this family are co-purified with metal ions, they still require the exogenous supply of metal ions for full activation. However, the specific roles these metal ions play during catalysis are yet to be well understood. Here, we report the metal ion requirement for phosphatase activities of S. aureus Cap8C and L. rhamnosus Wzb. AlphaFold-predicted structures of the two PTPs suggest that they are members of the PHP family. Like other PHP phosphatases, the two enzymes have a catalytic preference for Mn²⁺, Co²⁺ and Ni²⁺ ions. Cap8C and Wzb show an unusual thermophilic property with optimum activities over 75 °C. Consistent with this model, the activity–temperature profiles of the two enzymes are dependent on the divalent metal ion activating the enzyme.

To search for novel thermostable esterase for optimized industrial applications, esterase from a thermophilic eubacterium species, Thermoanaerobacter tengcongensis MB4 was purified and characterised in this work. Sequence analysis of T. tengcongensis esterase with other homologous esterases of the same family revealed an apparent tail at the C-terminal that is not conserved across the esterase family. Hence, it was hypothesized that the tail is unlikely to have an essential structural or catalytic role. However, there is no documented report of any role for this tail region. We probed the role of the C-terminal domain on the catalytic activity and substrate preference of Thermoanaerobacter tengcongensis esterase EstA3 with a view to see how it could be engineered for enhanced properties. To achieve this, we cloned, expressed, and purified the wild-type and the truncated version of the enzyme. In addition, a naturally occurring member of the family (from B. brevis) that lacks the C-terminal tail was also made. In vitro characterization of the purified enzymes showed that the C-terminal domain contributes significantly to the catalytic activity and distinct substrate preference of T. tengcongensis esterase EstA3. All the three recombinant enzymes showed highest preference to paranitrophenyl butyrate (pNPC4), which suggests they are true esterases, not lipases. kinetic data revealed that truncation had a slight effect on the substrate-binding affinity. Thus, the drop in preference towards long-chain substrates might not be a result of substrate binding affinity alone.

Protein tyrosine phosphatases (PTPs) of the polymerase and histidinol phosphatases (PHP) superfamily with characteristic phosphatase activity dependent on divalent metal ions are found in many gram-positive bacteria. Although members of this family are co-purified with metal ions, they still require the exogenous supply of metal ions for full activation. However, the specific roles these metal ions play during catalysis are yet to be well understood. Here, we report the metal ion requirement for phosphatase activities of S. aureus Cap8C and L. rhamnosus Wzb. AlphaFold-predicted structures of the two PTPs suggest that they are members of the PHP family. Like other PHP phosphatases, the two enzymes have catalytic preference for Mn2+, Co2+, and Ni2+ ions. Cap8C and Wzb show an unusual thermophilic property with optimum activities over 75oC. Pre-incubation experiments show that the two enzymes are not inherently thermostable suggesting that certain catalytic step(s) in the reaction mechanism has a high activation energy. Consistent with this model, the activity-temperature profiles of the two enzymes are dependent on the divalent metal ion activating the enzyme.

G-quadruplex DNAzymes are short DNA aptamers with repeating G4 quartets bound in a non-covalent complex with hemin. These G4/Hemin structures exhibit versatile peroxidase-like catalytic activity with a wide range of potential applications in biosensing and biotechnology. Current efforts are aimed at gaining a better understanding of the molecular mechanism of DNAzyme catalysis as well as devising strategies for improving their catalytic efficiency. Multimerisation of discrete units of G-quadruplexes to form multivalent DNAzyes is an emerging design strategy aimed at enhancing the peroxidase activities of DNAzymes. While this approach holds promise of generating more active multivalent G-quadruplex DNAzymes, few examples have been studied and it is not clear what factors determine the enhancement of catalytic activities of multimeric DNAzymes. In this study, we report the design and characterisation of multimers of five G-quadruplex sequences (AS1411, Bcl-2, c-MYC, PS5.M and PS2.M). Our results show that multimerisation of G-quadruplexes that form parallel structure (AS1411, Bcl-2, c-MYC) leads to significant rate enhancements characteristic of cooperative and/or synergistic interactions between the monomeric units. In contrast, multimerisation of DNA sequences that form non-parallel structures (PS5.M and PS2.M) did not exhibit similar levels of synergistic increase in activities. These results show that design of multivalent G4/Hemin structures could lead to a new set of versatile and efficient DNAzymes with enhanced capacity to catalyse peroxidase-mimic reactions.

The roles of Mg ²⁺ and Zn ²⁺ ions in promoting phosphoryl transfer catalysed by alkaline phosphatase are yet to be fully characterised. We investigated the divalent metal ion requirements for the monoesterase and diesterase activities of calf intestinal alkaline phosphatase. The synergistic effect of Mg ²⁺ and Zn ²⁺ in promoting the hydrolysis of para-nitrophenyl phosphate (monoesterase reaction) by alkaline phosphatase is not observed in the hydrolysis of the diesterase substrate, bis-para-nitrophenyl phosphate. Indeed, the diesterase reaction is inhibited by concentrations of Mg ²⁺ that were optimal for the monoesterase reaction. This study reveals that the substrate specificities of alkaline phosphatases and related bimetalloenzymes are subject to regulation by changes in the nature and availability of cofactors, and the different cofactor requirements of the monoesterase and diesterase reactions of mammalian alkaline phosphatases could have significance for the biological functions of the enzymes.