(681b) High Throughput, Mutliplexed Detection of miRNA Analogs Using Micellar Electrokinetic Chromatography

Short, non-coding micro-RNAs (miRNA) have been implicated in an increasing number of cancers, and miRNA detection has emerged as a promising means of early-stage cancer diagnosis. miRNA are present in extremely low (attomole) levels in cell extracts, making them difficult and time-consuming to detect by conventional means. miRNA are only 22 nucleotides in length, and therefore make marginally stable duplexes with complementary oligonucleotide probes. Complicating matters even further is the observation that many miRNA differ by only a single base in sequence. Available methods, such as microchip arrays and bead-based assays, suffer from poor reproducibility and false-positive readings. Most implementations also require a PCR-type amplification step that presents inefficiencies given the short template length.

Here, we present a fast, specific, high-sensitivity miRNA detection method operating in a capillary electrophoresis (CE) format. Detection relies on the hybridization-based linkage of a surfactant micelle and a dsDNA oligomer pre-loaded with a flurorescent intercalating dye (YOYO-1) in the presence of target miRNA. In so doing, the electrophoretic mobility of the YOYO-1 DNA is shifted into a detection zone of the CE electropherogram. High-affinity, high-selectivity miRNA binding is ensured by use of synthetic DNA analogues (γ-substituted PNAs). The YOYO-1 DNA may also be made highly fluorescent by simply extending its length as required.

We will discuss our recent efforts to implement the method for detection of the let-7 series of miRNA. Preliminary results show that up to 13 targets are reliably identified in a 5-minute run with nanomolar sensitivity. We find that high selectivity, over a wide range of single-base mismatches, is realized for runs performed at slightly elevated temperature (40-45°C). We also discuss recent efforts to increase sensitivity by orders of magnitude using stacking and sweeping methods in inhomogeneous buffer systems.