Promiscuous dye binding by a light-up aptamer: application for label-free multi-wavelength biosensing.

Light-up DNA aptamers are promising label-free signal-transducers for biosensing applications due to their high chemical stability and low synthetic cost. Herein, we demonstrate that a dapoxyl DNA aptamer DAP-10-42 can be converted into a sensor generating a fluorescence signal at different wavelengths in the range of 500-660 nm depending on the dye that is present. This results from the discovered promiscuity of DAP-10-42 in binding fluorogenic dyes including arylmethane dyes. We have designed a split DAP-10-42 aptasensor for the detection of a katG gene fragment from Mycobacterium tuberculosis with a point mutation causing isoniazid resistance. Efficient interrogation of the gene fragment after nucleic acid sequence-based amplification (NASBA) is achieved directly in a protein-containing NASBA sample. This report lays a foundation for the application of the DAP-10-42 aptamer as a versatile sensing platform.

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

A visual cascade detection system has been applied to the detection and analysis of drug-resistance profile of Mycobacterium tuberculosis complex (MTC), a causative agent of tuberculosis. The cascade system utilizes highly selective split RNA-cleaving deoxyribozyme (sDz) sensors. When activated by a complementary nucleic acid, sDz releases the peroxidase-like deoxyribozyme apoenzyme, which, in complex with a hemin cofactor, catalyzes the color change of the sample's solution. The excellent selectivity of the cascade has allowed for the detection of point mutations in the sequences of the MTC rpoB, katG, and gyrA genes, which are responsible for resistance to rifampin, isoniazid, and fluoroquinolone, respectively. When combined with isothermal nucleic acid sequence based amplification (NASBA), the assay was able to detect amplicons of 16S rRNA and katG mRNA generated from 0.1 pg and 10 pg total RNA taken for NASBA, respectively, in less than 2 h, producing a signal detectable with the naked eye. The proposed assay may become a prototype for point-of-care diagnosis of drug resistant bacteria with visual signal output.

Toward a Rational Approach to Design Split G-Quadruplex Probes.

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, H2O2-mediated 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.

Label-Free Pathogen Detection by a Deoxyribozyme Cascade with Visual Signal Readout.

A colorimetric nucleic acid based test for label-free pathogen detection has been developed and used for the detection of the Zika virus. The test relies on nucleic acid sequence-based amplification (NASBA) of a viral RNA followed by interrogation of the amplicon by a cascade of deoxyribozymes constituting a visual split deoxyribozyme (vsDz) probe. The probe consists of a split phosphodiesterase deoxyribozyme, which forms its catalytic core upon binding to a specific amplicon fragment. The catalytically active complex recognizes and cleaves an inhibited peroxidase-like deoxyribozyme (PDz), thereby activating it. Active PDz catalyzes hydrogen peroxide-mediated oxidation of a colorless substrate into a colored product, thereby generating a visible signal. Viral RNA (106 copies/mL or higher) triggers intense color within 2 hr. The test selectively differentiates between Zika and closely related dengue and West Nile viruses. The reported technology combines isothermal amplification and visual detection and therefore represents a basis for the future development of a cost-efficient and instrument-free method for point-of-care nucleic acid analysis.

Alphanumerical Visual Display Made of DNA Logic Gates for Drug Susceptibility Testing of Pathogens.

Molecular diagnostics of drug-resistant pathogens require the analysis of point mutations in bacterial or viral genomes, which is usually performed by trained professionals and/or by sophisticated computer algorithms. We have developed a DNA-based logic system that autonomously analyzes mutations found in the genome of Mycobacterium tuberculosis complex (MTC) bacteria and communicates the output to a human user as alphanumeric characters read by the naked eye. The five-gate system displays "O" ("no infection") for the absence of MTC infection and "P" or "F" for passing or failing a drug-susceptibility test, respectively.