Peptide nucleic acids (PNAs) are synthetic DNA analogs with superior DNA and RNA binding properties. PNAs have the phosphodiester backbone of natural nucleic acids replaced by a N-(2-amino-ethyl)-glycine version, which aids in binding stability but also leads to poor solubility and a tendency to aggregate in aqueous buffers. Maintaining the activity of PNAs in biosensing platforms has been a challenge in their wider implementation. One means to control the presentation of these powerful but poorly soluble probes is to end-alkylate them for their organized assembly into surfactant microstructures, including micelles of nonionic surfactant, liposomes, or supported lipid bilayers, depending on the mode of detection desired.<br/><br/>Here we present the molecular design of a self-assembled PNA amphiphile system designed to detect sub-femtomolar amounts of miRNA in capillary electrophoresis.[1] A sandwich-type binding scheme is utilized that links a surfactant micelle with a long, dye infused length of DNA when a specific miRNA target is present. The electrophoretic mobility of the complex is set by the length of DNA attached to it, and multiple targets are detected in the same run by employing distinct lengths of DNA for each target. Additionally, use of long DNA tags provides ultra-high levels of fluorescence when infused with intercalating dyes such as YOYO-1 or PicoGreen. We demonstrate the stability and single-base-mismatch sensitivity of the sandwich hybridization assay in capillary electrophoresis, where 10-20 different targets are resolved in a 5-minute separation window. We also discuss implementation of a next-generation PNA design (γPNA) which has a mini-PEG substitution at the γ position of the PNA backbone, leading to very high binding stability and an ability to specifically bind 5-6 base sequences at room temperature. We will also discuss efforts to improve detection limits by field-focusing methods and micelle-based separations compatible with kilobase-length DNA.<br/><br/>[1]Goldman et. al. Biomacromolecules, 2013, 14(7):2253-61.

Molecular design of peptide nucleic acid amphiphiles as self-assembled probes for electrophoretic miRNA detection

249th National Meeting (2015)

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