Research Project
9620476
ABSTRACT To date, more than 1000 microRNAs (miRs) have been identified in humans. Aberrant miRs expression has been associated with many human diseases, and especially with cancer. Therefore, early detection and quantitation of those miRs can have a significant impact on patients' outcome and survival rate. The utility of miRs as potential clinical and diagnostic tools is severely limited by the availability of simple and robust methods for their detection and quantitation in biological samples. Novelty and significance of this proposal is based on the development of ZATA?s fluorescent-labeled new generation oligonucleotide (ON) based diagnostic probes (Z-Probes) that will enable the detection and quantification of several target miRs simultaneously in a single sample. Ability to simultaneously detect several target miRs, along with other advantages, such as simplicity of procedure, high sensitivity, selectivity, reproducibility, makes our method superior to currently used approaches, such as qPCR. The second and equally important application of Z-probes is for detection and quantitation of synthetic ONs in PK/PD studies of ONs-based therapeutics, that are widely tested against cancer, microorganism mediated, genetic and other diseases. Z-probes take an advantage on ZATA?s novel ONs with controlled charge neutralization of their backbones. Our preliminary data strongly supports the feasibility of Z-probes development into diagnostic modalities for research and clinical use. As preliminary results we synthesized and characterized several model Z-probes and used them for the detection and quantification of the synthetic natural and modified RNA in buffers and in serum. Obtained data have demonstrated high feasibility of the proposed probes for diagnosis purpose. In the framework of this application, we anticipate achieving several milestones that will validate our Z-probes as a diagnostic modality for research and clinical studies. Those milestones are: (1) Use of Z-probes for quantification of short interference RNAs (siRNA), antisense ONs, and synthetic analogs of miRs in biological samples (tissue, whole blood, plasma, cell culture), and in urine; (2) Demonstration of the sequence-specificity and high selectivity of Z-probes by testing them against non-complimentary and mismatched target nucleic acids; (3) Scaling-up and optimizing of the method for synthesis of Z-probes; (4) Use of Z-probes for the quantification of endogenous miRs in the cell cultures, as well as in human serum, blood and tissue samples. The outcome of this Phase I will be methods and technical protocols for the detection and quantification of antisense oligonucleotides, siRNAs, and miRs in biological samples using Z- probes. Method of synthesis of Z-probes and composition of matter are claimed in new national stage patent applications in US, EU, Canada, and Australia.
