Split Dapoxyl Aptamer for Sequence-Selective Analysis of Nucleic Acid Sequence Based Amplification Amplicons

Abstract

Hybridization probes have been used for the detection of single nucleotide variations (SNV) in DNA and RNA sequences in the mix-and-read formats. Among the most conventional are Taqman probes, which require expensive quantitative polymerase chain reaction (qPCR) instruments with melting capabilities. More affordable isothermal amplification format requires hybridization probes that can selectively detect SNVs isothermally. Here we designed a split DNA aptamer (SDA) hybridization probe based on a recently reported DNA sequence that binds a dapoxyl dye and increases its fluorescence (Kato, T.; Shimada, I.; Kimura, R.; Hyuga, M., Light-up fluorophore-DNA aptamer pair for label-free turn-on aptamer sensors. Chem. Commun. 2016, 52, 4041−4044). SDA uses two DNA strands that have low affinity to the dapoxyl dye unless hybridized to abutting positions at a specific analyte and form a dye-binding site, which is accompanied by up to a 120-fold increase in fluorescence. SDA differentiates SNV in the inhA gene of Mycobacterium tuberculosis at ambient temperatures and detects a conserved region of the Zika virus after isothermal nucleic acid sequence based amplification (NASBA) reaction. The approach reported here can be used for detection of isothermal amplification products in the mix-and-read format as an alternative to qPCR.

Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics

“…These strands have low affinity to the dapoxyl dye unless specific targets bind to adjacent positions and form binding sites for the dye to generate fluorescent signals. In this work, they used these strands to successfully distinguish a fragment of the inhA gene of Mycobacterium tuberculosis from its single-base variant and detect 10 nM of Z-147 RNAs (a fragment of the ZIKV envelope gene) extracted using Trizol LS ( Kikuchi et al, 2019 ). With portable fluorometers, this probe has the potential to achieve POC diagnosis.…”

Investigation of the interaction between split aptamer and vascular endothelial growth factor 165 using single molecule force spectroscopy

“…Dynamics parameters on the dissociation process of split aptamer/VEGF 165 complex, such as the binding lifetime τ (=1/k off ), k off , and x β , was estimated according to Equation (1). As shown in It demonstrated that split aptamer of VEGF 165 still exhibited high affinity to its target.…”

“…Split aptamer was obtained by dividing the intact aptamer, which could self‐assemble to produce a positive signal when the target was present. They had a good enhancement effect in enhancing signal‐to‐background ratio and reducing nonspecific signal because of their excellent merit in avoiding steric hindrance. Thus, the application of split aptamer was various, such as biosensing, bioimaging, drug delivery, and theranostics …”

Short Duplex Module Coupled to G-Quadruplexes Increases Fluorescence of Synthetic GFP Chromophore Analogues

“…Such RNA-based signaling elements are present in malachite green/RNA aptamer pair [15,16] and GFP chromophore analogue DFHBI/split spinach aptamer pair [17,18]. Despite increased chemical and biological stability of DNA aptamers compared to RNA ones, the only example of such a biosensor based on light-up dye-DNA aptamer pair as "signaling element" is dapoxyl dye/DAP-10-42 for thrombin and ATP detection [19] and "split" DAP-10 variant for nucleic acids analysis [20]. Several examples are devoted to label-free sensors utilizing G-quadruplex DNAs and their light-up ligands as "signaling elements", including zinc(II)-protoporphyrin IX [21], thioflavin T [22], iridium(III) complexes [23] and crystal violet [24].…”