Toward a Rational Approach to Design Split G-Quadruplex Probes

Abstract

Hybridization probes have become an indispensable tool for nucleic acid analysis. Systematic efforts in probe optimization resulted in their improved binding affinity, turn-on ratios, and ability to discriminate single nucleotide substitutions (SNSs). The use of split (or multicomponent) probes is a promising strategy to improve probe selectivity and enable an analysis of folded analytes. Here, we developed criteria for the rational design of a split G-quadruplex (G4) peroxidase-like deoxyribozyme (sPDz) probe that provides a visual output signal. The sPDz probe consists of two DNA strands that hybridize to the abutting positions of a DNA/RNA target and form a G4 structure catalyzing, in the presence of a hemin cofactor, H 2 O 2mediated oxidation of organic compounds into their colored oxidation products. We have demonstrated that probe design becomes complicated in the case of target sequences containing clusters (two or more) of cytosine residues and developed strategies to overcome the challenges to achieving high signal-to-noise and excellent SNS discrimination. Specifically, to improve selectivity, a conformational constraint that stabilizes the probe's dissociated state is beneficial. If the signal intensity is compromised, introduction of flexible non-nucleotide linkers between the G4-forming and target-recognizing elements of the probe helps to decrease the steric hindrance for G4 PDz formation observed as a signal increase. Varying the modes of G4 core splitting is another instrument for the optimal sPDz design. The suggested algorithm was successfully utilized for the design of the sPDz probe interrogating a fragment of the Influenza A virus genome (subtype H1N1), which can be of practical use for flu diagnostics and surveillance.

Towards Point of Care Diagnostics: Visual Detection of Meningitis Pathogens Directly from Cerebrospinal Fluid

“…Despite the presence of nonspecific amplification products, the negative control samples did not produce appreciable color change (Figure 3A, sample 1). Indeed, we and others have earlier demonstrated that the biPxD probes have exceptional selectivity enabling differentiation of single nucleotide substitutions (SNP) in nucleic acids even at room temperature [20,22,23,36] . Therefore, the approach described here can potentially enable detection of point mutations for determination of drug resistance and genotyping bacteria, which will be demonstrated in the follow up studies.…”

“…Extra nucleotides non‐complementary to the analyte were added to both strand m and f (shown in low cases in Figure 2A). These nucleotides were required to fold the stands in stem‐loop structures (Figure 2C) and reduce their associations in G‐4 in the absence of analytes [36] . Probes with strands lacking intramolecular folding produced high background color change in the absence of analytes (data not shown).…”

“…Indeed, we and others have earlier demonstrated that the biPxD probes have exceptional selectivity enabling differentiation of single nucleotide substitutions (SNP) in nucleic acids even at room temperature. [20,22,23,36] Therefore, the approach described here can potentially enable detection of point mutations for determination of drug resistance and genotyping bacteria, which will be demonstrated in the follow up studies. We hope that further development of the proposed technology may enable tests for diagnosis of meningitis infections with visual output signal suitable for expedite determination of meningitis pathogens even in low-resource hospitals.…”

“…[35] However, this approach is applicable only for fluorescent probes with LOD down to 10 pM. LOD demonstrated by biPxD sensors is about 60 nM, [36] unless additional amplification steps are added. [37] Therefore, conversion of dsDNA to ssDNA was a required step to achieve efficient probe-analyte complex formation.…”

Cascade of deoxyribozymes for the colorimetric analysis of drug resistance in Mycobacterium tuberculosis

“…S3). The discriminatory ability of the split probes can be improved via one of the two approaches (Connelly et al, 2019). In the first approach, the target-binding fragment of strand S was shortened to form strand S2 (Fig.…”