Multicomponent hybridization probe with a label-free reporter based on a DNA light-up aptamer

AnnaMarie Knowles, Yulia Gerasimova

Chemistry Department, University of Central Florida

Multicomponent probes offer some advantages in nucleic acid analysis over conventionally used monolith probes, such as Molecular Beacon probe. Such advantages include selectivity in nucleic acid recognition over wider conditions, and better accessibility and more efficient binding to structured nucleic acid targets. In addition, to report the presence of a nucleic acid target, conventional probes usually rely on fluorescent signal, for which the probe (or signal reporter) needs to be covalently linked to a fluorophore and/or quencher, which increases the costs associated with the probe optimization and assay development. In this work we aim at designing and testing a label-free multicomponent probe that takes advantage of a DNA light-up aptamer DAP-10, which was reported to bind and increase fluorescence of fluorogenic dapoxyl sulfonyl dyes.¹ We have previously demonstrated the ability of DAP-10-42 to increase fluorescence of other dyes (e.g. auramine O).² In order to use DAP-10-42 as a reporter for multicomponent probes, it was split into two strands, which were also elongated with the sequences complementary to each other, at one end, and with the target-binding sequences, at the opposite end. Such splitting prevented formation of the dye-binding pocket unless the target was present. Therefore, the target’s presence can be reported via an increase of the fluorogenic dye (auramine O) bound to the DAP-10-derived reporter module. As a model target, we used miRNA92a which promotes cell proliferation, migration and survival by directly targeting the tumor suppressor

gene NF2 in colorectal and lung cancer cells.³ We tested two different approaches to DAP-10 splitting, based on our prior work with split DAP-10² and on the approach utilized by another group.⁴ We compared the two designs in terms of kinetics of the signal generation upon target addition, as well as in term of limit of detection. Our data can assist in designing multicomponent probes with DAP-10-based signal reporting system for sequence-specific detection of nucleic acid targets of interest.

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