Universal Drag Tag for Direct Quantitative Analysis of Multiple MicroRNAs

Using microRNA (miRNA) as molecular markers of diseases requires a method for accurate measurement of multiple miRNAs in biological samples. Direct quantitative analysis of multiple miRNAs (DQAMmiR) has been recently developed based on a classical hybridization approach. In DQAMmiR, miRNAs are hybridized with excess fluorescently labeled complementary DNA probes. Capillary electrophoresis (CE) is used to separate the unreacted probes from the hybrids and the hybrids from each other. The challenging separation is achieved by using two types of mobility modifiers. Single-strand DNA binding protein (SSB) is added to the running buffer to bind and shift the single-stranded unreacted probes from the double-stranded hybrids. Different drag tags are built into the probes to introduce significant differential mobility between their respective hybrids. For the method to be practical it requires a universal extendable drag tag. Polymers are a logical choice for making extendable drag tags. Our recent theoretical work suggested that short peptides could provide a sufficient mobility shift to facilitate DQAMmiR. Here, we experimentally confirm this prediction in the analysis of five miRNAs: mir10b, mir21, mir125b, mir145, and mir155. We conjugated four fluorescently labeled DNA molecules with peptides of 5, 10, 15, or 20 neutral amino acids in length; the fifth probe was peptide-free. The peptide tags showed no interference with SSB binding to the probes and facilitated separation of the five hybrids. The mobilities of the five hybrids were used to refine the previously suggested theory. The analysis was performed in both a pure buffer and in cell lysate. Our analysis of the experimental data suggests that using DNA peptide probes can readily facilitate simultaneous analysis of more than 10 miRNAs.