AUTOLIGATION PROBES FOR HIGHLY SELECTIVE RNA IMAGING

Stanford University, Stanford CA

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): We propose to develop new, highly selective chemical methods for detecting, identifying, and imaging RNAs. Our approach makes use of a simple nucleophilic displacement that leads to ligation of two short DNA fragments. This reaction is highly sequence selective. The method can be used in solution, on beads and arrays, and in cellular preparations. The modified probes are easily prepared directly on a DNA synthesizer using automated methods, and the reaction itself requires neither enzymes nor even any added reagents beyond the probes themselves. This simplicity makes it possible to carry out the reaction even in living cells. Our preliminary results have shown that this "autoligation" reaction can be linked to fluorescence color-change strategies for direct reporting on the sensing of RNA sequences. We have recently developed a highly efficient quencher-based approach in which the fluorescence quencher is also the leaving group in the ligation. The resulting probes "light up" when an RNA is sensed. Recent work has shown that these QUAL probes can sense RNAs in living bacterial cells, and show single nucleotide specificity. This has not been possible to achieve before. In the long term we hope to develop autoligation probes for clinical application. We believe they may one day be useful in rapid and accurate detection and identification of pathogenic bacteria strains in clinical samples, for imaging of disease-related RNAs in human tissue specimens, and finally, for molecular imaging of tissues in living human patients. In the short term covered by this proposal, our specific aims are: (1) New designs for quenched probes (2) Autoligation in signal amplification strategies (3) Universal arrays for detection of RNAs and DNAs from blood and tissues (4) Sensing bacterial RNAs (5) Imaging RNAs in human cells.