Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.
miRNA is a promising class of biomarkers whose levels can be assayed to detect various forms of cancer and other serious diseases. These short, noncoding nucleic acids are difficult to detect due to their low abundance and the marginal stability of their duplexes with DNA probes. In addition, miRNAs within the same family have high sequence homology, and often, related miRNA differ in sequence by only a single base. In this report, we demonstrate an independent detection seven members of the let‐7 family of miRNA in a single run. Key to success is the use of mini‐PEG‐substituted PNA amphiphiles (γPNAA) and highly fluorescent DNA nanotags in micelle tagging electrophoresis (MTE). Multiplexed detection is accomplished in capillary electrophoresis (CE) using oligomeric nanotags of pre‐programmed lengths where the presence of a specific miRNA links its nanotag to a micelle drag‐tag, which shifts the nanotag elution time to a defined region for detection. We further demonstrate that the peak shape and elution time are unaffected by the presence of up to 10 mg/ml of serum protein in the sample, with a total runtime of less than 4 min and a LOD of 10–100 pM. We discuss efforts to substantially decrease the detection limit using nanotags that are >1000 bp in length.
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