DNA Nanotechnology: The Development of Multi-Functional Hybridization Sensors

Abstract

Hybridization probes have been used to detect specific nucleic acids for the last 50 years. These probes have medical applications, including identifying disease-causing genes or multi-drug resistant bacteria. To be considered robust, a probe should have high selectivity at ambient or low temperatures, be able to detect folded analytes, and remain economical for use in clinical settings. This work will uncover a challenge faced by molecular beacon probes (MBP), describe an adaptation to MBPs that enables the hybridization of the probe to a folded target, a multicomponent DNA sensor (OWL2) that overcomes common challenges faced by hybridization probes, and a thresholding sensor (MB-Th) that allows for the quantification of microRNA. Using ssDNA segments, the MBP adaptation and OWL2 sensor are able to hybridize with and detect folded analytes. The OWL2 sensor contains two analyte-binding arms to unwind folded analytes and two sequence-specific strands that bind both the analyte and a universal molecular beacon (UMB) probe to form a fluorescent ‘OWL’ structure. The sensor can differentiate single base mismatches in folded analytes in the temperature range of 5–38 °C, even when challenged with excess wild-type analytes. The MB-Th sensor consists of two gates with increasing affinity for the target, with each varying in thermodynamic stability. The gates bind to separate molecular beacons, each with a unique fluorophore, and produce distinct signals that can be measured simultaneously. Both sensor designs are cost-efficient since the same UMB probe can be used to detect any analyte sequence. These sensors have significant clinical benefits for diagnosing non-invasive early-stage cancer and cancers associated with miRNA dysregulation.