We propose to develop new, highly selective chemical methods for detecting, identifying, and imaging RNAs in cells by fluorescence signals. Our approach makes use of an RNA-templated fluorogenic reaction between two modified DNA probes. This reaction is highly sequence selective, and is able to discriminate even between single nucleotide variants of a genetic target. The approach is versatile, and can be used in solution, on beads and arrays, in fixed tissues, and in live bacterial and human cells. The modified probes studied to date are easily prepared directly on a DNA synthesizer using automated methods, and the reaction itself requires no enzymes nor even any added reagents beyond the probes themselves. Such an approach offers considerable benefits in cost, speed, and simplicity over current genetic identification methods such as PCR combined with sequencing, and these advantages should make possible some potentially life-saving tests that are not feasible now. During the recent funding period we have made strong progress toward our long-term goals. Among the advances reported recently are the development of a new fluorogenic "light up" strategy for engendering signal;a number of new multicolor labeling and signaling strategies;new improved nucleophiles that speed reaction and improve signal/noise;the first applications of RNA- templated chemistry in bacterial and human cells;and the first detection of single nucleotide differences directly in living cells. In the long term we aim to develop RNA-templated probes for clinical application and for widespread use in biomedical research. We believe that such probes may one day be useful in rapid and accurate quantitation of RNAs in studies of gene expression, for detection and identification of pathogenic bacteria strains in clinical samples, for identifying disease-related RNAs in human tissue specimens, and one day, for imaging molecular genetics of surface tissues in human patients. In the near term covered by this proposal, we are focusing on improvements to signal-to-noise that are needed for targeting disease-related cellular mRNAs.
Our specific aims i nclude (1) study of new strategies for reducing background signal;(2) development of new bioorthogonal fluorogenic reactions;and (3) collaborative development and testing of probes for diagnosing and monitoring hematologic diseases. We propose to develop new, highly selective chemical methods for detecting, identifying, and imaging RNAs in cells by fluorescence signals. Our approach makes use of pairs of DNA probes that bind adjacently on RNA in cells and react with one another to produce a visible signal. These probes will be useful as biomedically important tools for research and clinical laboratories, allowing rapid and inexpensive identification of disease-related genetic mutations and variations.
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