Quantitative Analysis of Locked Nucleic Acid and DNA Competitive Displacement Events on Microspheres.

Synthetic analogues of natural oligonucleotides known as locked nucleic acids (LNAs) offer superior nuclease resistance and cytocompatibility for numerous scenarios ranging from detection to intracellular imaging of nucleic acids. While recognized as stronger hybridization partners than equivalent DNA residues, quantitative analysis of LNA hybridization activity is lacking, especially with respect to competitive displacement of the original hybridization partner by another oligonucleotide. In the current study, we perform measurements of toehold-mediated competitive displacement of soluble, fluorescently labeled primary targets from probe strands immobilized on microspheres using high throughput flow cytometry.

Deep-tissue optical imaging of near cellular-sized features.

Detection of biological features at the cellular level with sufficient sensitivity in complex tissue remains a major challenge. To appreciate this challenge, this would require finding tens to hundreds of cells (a 0.1 mm tumor has ~125 cells), out of ~37 trillion cells in the human body.

Analysis of in Situ LNA and DNA Hybridization Events on Microspheres.

The hybridization activity of single-stranded DNA and locked nucleic acid (LNA) sequences on microspheres is quantified in situ using flow cytometry. In contrast to conventional sample preparation for flow cytometry that involves several wash steps for posthybridization analysis, the current work entails directly monitoring hybridization events as they occur between oligonucleotide-functionalized microspheres and fluorescently tagged 9 or 15 base-long targets. We find that the extent of hybridization between single-stranded, immobilized probes and soluble targets generally increases with target sequence length or with the incorporation of LNA nucleotides in one or both oligonucleotide strands involved in duplex formation.

Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation.

Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins.