Peroxidase-like Activity of G-Quadruplex/Hemin Complexes for Colorimetric Nucleic Acid Analysis: Loop and Flanking Sequences Affect Signal Intensity

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Background/Objectives: Some G-quadruplex (G4)-forming nucleic acid sequences bind a hemin cofactor to enhance its peroxidase-like activity. This has been implemented in a variety of bioanalytical assays benefiting from analyte-dependent peroxidation of a chromogenic organic substrate (e.g., ABTS) to produce a color change. Adenine and cytosine nucleotides in the vicinity of the G4 hemin-binding site promote the peroxidation reaction. In this work, the effect of G4 loop and flanking nucleotides on the colorimetric signal of split hybridization probes utilizing hemin-G4 signal reporters was tested. Methods: G4s varying by loop sequences and flanking nucleotides were tested with hemin for ABTS peroxidation (A420), and the signal was compared with that produced by the most catalytically efficient complexes reported in the literature using one-way ANOVA and post hoc pairwise comparison with Tukey’s HSD test. The best G4s were used as signal transducers in the split peroxidase deoxyribozyme (sPDz) probes for sensing two model nucleic acid analytes, as well as in a cascade system, where the analyte-dependent assembly of an RNA-cleaving deoxyribozyme 10–23 results in G4 release. Results: Intramolecular G4s (G3T)3G3TC or G3T3G3ATTG3T3G3 were found to be the most efficient hemin PDzs. When splitting intramolecular G4 for the purpose of sPDz probe design, the addition of a flanking d(TC) sequence at one of the G4 halves or d(ATT) in a loop connecting the second and third G-tracts helps boost analyte-dependent signal intensity. However, for the cascade system, the effect of d(TC) or d(ATT) in the released G4 was not fully consistent with the data reported for intramolecular G4-hemin complexes. Conclusions: Our findings offer guidance on the design of split hybridization probes utilizing the peroxidase-like activity of G4-hemin complexes as a signal transducer.

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