Talk abstracts

Talk on Saturday 11:30-11:45am submitted by Munira Fouz

Enhanced MicroRNA Detection Assays Based on Tyramide Signal Amplification and GammaPNA Probes.

Munira Fouz (Department of Chemistry, Carnegie Mellon University), Lauren Xu (Department of Chemistry, Carnegie Mellon University), Danith Ly (Department of Chemistry, Carnegie Mellon University), Veronica Hinman (Department of biological sciences, Carnegie Mellon University), Subha R. Das (Department of Chemistry, Carnegie Mellon University), Bruce Armitage (Department of Chemistry, Carnegie Mellon University)

The ability to detect spatial and temporal microRNA (miRNA) expression and distribution at different stages of growth cycles are essential for understanding the biological roles of miRNA and miRNA-associated gene regulatory networks. The success of detection methods such as in situ hybridization depends on the stability of the double helix formed by the target and the probe, especially for the detection of shorter RNA sequences like miRNA. Peptide nucleic acids (PNAs) installed with a relatively small, hydrophilic entity at the γ-position of the backbone, i.e. γPNAs, make high affinity probes. Miniprobes, which are short γPNAs designed by splitting a long probe sequence, can recognize a target sequence by hybridizing adjacent to each other with high cooperative affinity. These miniprobes also show high sequence specificity with significant destabilization of duplexes for non-target sequences bearing single mismatch nucleotides. Here we develop enhanced fluorescent detection tools combining the special features of gammaPNA mini-probes and Tyramide Signal Amplification (TSA) technology for detection and quantification of miRNA. The miniprobe design allows incorporation of functional groups for enhanced amplification, improving signal-to-noise ratio. We currently employ these tools to understand the regulatory mechanisms of cell specification during early development of sea star larva; a useful model system to study tissue regeneration. In addition, the approach can be readily generalized to any RNA target.

Keywords: Gamma-modified Peptide Nucleic Acids (PNA), Regenerative biology, Tyramide Signal Amplification (TSA)