No abstract

DNA-DNA reactions can be monitored with a label-free fluorogenic reaction. Guanosine-rich, single-stranded DNA oligonucleotides bind to thioflavin-T (ThT) and enhance the fluorescence of the dye. We discovered a novel DNA sequence that produces fluorescence upon binding to ThT. We denote this oligonucleotide ThTSignal. We use ThTSignal as a label-free reporter for the activity of several designed DNA-DNA reactions (DNA circuits). The DNA circuits conditionally produce the ThTSignal oligonucleotide by association or by liberating the ThTSignal oligonucleotide from double-stranded DNA. This strategy offers label-free, cost-effective, fluorogenic detection of the molecular beacon reaction, split reporter reaction, one-step strand displacement reaction, and the entropy-driven amplifier reaction (a catalytic DNA circuit).

We selected an aptamer against a fluorogenic dye called Thioflavin T (ThT). Aptamers are single-stranded DNA that can bind a specific target. We selected the ThT aptamer using graphene oxide assisted SELEX and a low-cost Open qPCR instrument. We optimized, minimized, and characterized the best aptamer candidate against ThT. The aptamer, ThT dye, and the enzymatic strand displacement amplification (SDA) were used in a label-free approach to detect the micro RNA miR-215 in saliva and serum. The aptamer confers higher specificity than intercalating dyes but without expensive covalently modified DNA probes. This isothermal, low-cost, simple method can detect both DNA and RNA. The target, miR-215, was detected with a limit of detection of 2.6 nM.