Direct MicroRNA Detection Using Kilobase DNA Nantoags In Rapid Gel- Free Electrophoresis
The abnormal expression of microRNA (miRNA) within tissues has been linked to the onset of many diseases and cancers. The ability to employ miRNA as biomarkers for disease requires sensitive, high-throughput detection, ideally without expensive indirect enzymatic processing. We present our development of a direct, sensitive sandwich hybridization miRNA detection assay. We achieve hybridization of two probes to the short miRNA targets using high affinity PEG γ- carbon modified peptide nucleic acid amphiphiles (γPNAA). The γPNAA enables hybridization of a second, highly fluorescent DNA probe stained with intercalating dye, termed a nanotag, for sensitive detection of the low abundance miRNAs. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the hydrocarbon modification of the γPNAA with non-ionic surfactant micelles in a capillary electrophoresis running buffer, a technique known as micelle end-labeled free-solution electrophoresis (ELFSE). We demonstrate multiplexed detection of 6 let-7 miRNAs in 4 minutes, excellent selectivity against G-T wobble base-pair single base mismatches between let-7 miRNAs, and 100pM detection limits using our method. In an effort to increase detection sensitivity closer to those required for trace miRNA concentrations (fM), we have investigated the use of isotachophoretic injections and longer nanotags. Although longer nanotags can accommodate a greater amount of fluorescent dye for enhanced signal, they are a challenge to separate by micelle ELFSE. The longer DNA lengths overcome the additional friction of the transiently attached micelle and return to the inseparable freesolution limit. We find that n-alkyl polyoxyethylene ether surfactant (CiEj) wormlike micelles yield high drag forces on the electrophoresing DNAs, appearing to be ideal for large nanotag separations. However, micelle-micelle interaction at moderate concentrations leads to a background sieving network that reduces ELFSE separation efficiency for long DNAs. We propose new constitutive models that accurately capture the behavior of long DNA lengths in these micelle networks. We demonstrate optimization of surfactant buffers to achieve separation of 1-10 kilobase DNA in 3 minutes and increase DNA sequencing length of reads by refining the degree of micelle-micelle interaction. Finally, we demonstrate micelle ELFSE separations on a microchip format for dramatically reduced separation times.
- Chemical Engineering
- Doctor of Philosophy (PhD)