Quantitative Analysis of In Situ 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 in vitro 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 in situ measurements of toehold-mediated competitive displacement of soluble, fluorescently labeled primary targets from probe strands immobilized on microspheres using high throughput flow cytometry. Both LNA-DNA hybrid sequences and pure DNA sequences are employed as the immobilized strands, as soluble, fluorescently labeled 9-base-long primary targets, and as unlabeled 15-base-long secondary or competitive targets. In addition to comparing chemically substituted and unsubstituted sequences, we explore the effects of mismatched primary targets and the location of the toehold segment within the primary duplexes on the resulting displacement profiles. The primary duplex or double-stranded probe (dsprobe) systems implemented here exhibited varying responses to unlabeled secondary targets ranging from surprisingly modest primary target displacement activity despite the presence of a six base-long nucleotide toehold segment at the dsprobe free end to distinctive displacement profiles sensitive to LNA substitutions and the placement of the toehold segment closer to the microsphere surface.

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. Near-infrared optical imaging holds promise for high-resolution, deep-tissue imaging, but is limited by autofluorescence and scattering. To date, the maximum reported depth using second-window near-infrared (NIR-II: 1000-1700 nm) fluorophores is 3.2 cm through tissue. Here, we design an NIR-II imaging system, "Detection of Optically Luminescent Probes using Hyperspectral and diffuse Imaging in Near-infrared" (DOLPHIN), that resolves these challenges. DOLPHIN achieves the following: (i) resolution of probes through up to 8 cm of tissue phantom; (ii) identification of spectral and scattering signatures of tissues without a priori knowledge of background or autofluorescence; and (iii) 3D reconstruction of live whole animals. Notably, we demonstrate noninvasive real-time tracking of a 0.1 mm-sized fluorophore through the gastrointestinal tract of a living mouse, which is beyond the detection limit of current imaging modalities.

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. The rate constants for duplex formation, on the other hand, remain nearly identical for all but one probe-target sequence combination. The exception to this trend involves the LNA probe and shortest perfectly matched DNA target, which exhibit a rate constant that is an order of magnitude lower than any other probe-target pair, including a mismatched duplex case. Separate studies entailing brief heat treatments to suspensions generally do not consistently yield appreciable differences in associated target densities to probe-functionalized microspheres.

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. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.