There has been a growing concern in the medical and pharmaceutical industries regarding the increase in drug-resistant bacteria and viruses. Molecular adaptations, such as single nucleotide polymorphisms (SNPs) in genetic structure, can lead to unconventional protein folding rendering various drug therapies ineffective. Detection of these mutations is critical, and methods that can be used at the point of care settings are urgently needed. Recently, visual assays based on a split G-quadruplex (G4) peroxidase-like deoxyribozyme have been suggested for drug-susceptibility testing of bacterial pathogens. In these assays, the G-rich sequence of the deoxyribozyme is split into two subunits, which form a G4 structure only in the presence of a nucleic acid target. This structure forms a complex with hemin which catalyzes the hydrogen peroxide-mediated oxidation of an organic molecule (e.g. ABTS), producing a noticeable color change. Two strategies to split the four GGG repeats of the deoxyribozyme have been used – symmetrical splitting into the subunits containing two GGG repeats each, and asymmetrical splitting into 3:1 G-triplets. These two designs, while both effective in SNP differentiation, have never been directly compared under the same conditions. In this study, we used both symmetrical and asymmetrical approaches to design split peroxidase-like deoxyribozyme probes targeting the same fragment of the rpoB gene in Mycobacterium tuberculosis. We compared the detection limits and selectivity of both probes to find the best probe design to enable efficient and reliable differentiation of targets corresponding to rifampin-sensitive or resistant M. tuberculosis.

Visual split peroxidase-like deoxyribozyme probes for the detection of rifampin-resistance in Mycobacterium tuberculosis

255th National Meeting (2018)

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