Surface hybridization techniques, as widely practiced in DNA microarray and biosensor technologies, have penetrated into nearly every corner of fundamental and applied genomics. In these methods, the identity and/or the quantity of sample nucleic acid material are determined by its hybridization to complementary """"""""probe"""""""" molecules immobilized on a solid support. Despite their widespread application, demonstrated versatility, and powerful diagnostic capabilities, surface hybridization measurements are subject to physical and experimental complexities that can seriously confound interpretation of results. These complexities arise in part because the final diagnostic signal is a cumulative reflection of the history of multiple sequential, nonequilibrium rate processes including denaturation of target sample, slow competitive surface hybridization, and washing of the reacted surface to improve sequence discrimination. This research seeks to eliminate the central uncertainties in the practice of surface hybridization by developing diagnostic technology based on Morpholino, instead of DNA, probes. Morpholinos hybridize nucleic acids according to the usual base-pairing rules, possess excellent solubility and chemical stability, and can be prepared at lengths sufficient for nearly all diagnostic applications. Their lack of charge and unique hybridization properties promise a number of advantages which will be adapted to diagnostic applications through the following Specific Aims: 1). Critical comparison of Morpholino and DNA microarrays for pathogen detection in conventional fluorescence as well as label-free detection formats, 2). Development of fundamental understanding of the kinetics and thermodynamics of Morpholino surface hybridization, and 3). Establishment of optimized microarray assay protocols to minimize cross-hybridization and interference from sample secondary structure. Because Morpholinos are not charged, they hybridize with nucleic acids even under low salt conditions. Low ionic strength diagnostics promise to eliminate much of the measurement complexity by providing continually denaturing, high stringency conditions during the assay with prospects for abolishing the need for dedicated processing steps. Moreover, uncharged probes allow for highly specific electrostatic communication between the solid support and sample nucleic acids, a feature that will be exploited to develop electronic methods for controlling the hybridization reaction. By reassessing the nature of the probe platform, the proposed program seeks to overcome key challenges associated with today's large scale surface hybridization technologies, to make their data interpretation more transparent and robust, and ultimately to advance the practice of these powerful tools.
The goal of this program is to develop Morpholino probe technology for analysis of nucleic acids by surface hybridization, with applications to pathogen detection. The unique chemical properties of Morpholinos allow them to bind and thus identify nucleic acid sequences under low salt conditions where a markedly enhanced diagnostic response is realized compared to conventional approaches based on DNA probes, and where purely electronic (i.e. not requiring generation and measurement of light) detection of analyte and control of the assay are especially effective. Morpholino assays are expected to be highly suited to applications in gene expression and pathogen analysis, and a demonstration application aimed at identification of respiratorial pathogens is integrated as part of the project.
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