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 ZATAâs 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 ZATAâs 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.
Public Health Relevance Statement: NARRATIVE Development of the new generation of fluorescent-labeled charge-neutralized oligonucleotide (ON) probes for the detection and quantification of microRNAs and ON-based drug in biological samples by using ion- exchange HPLC with fluorescent detection is proposed. This method allows simultaneous and robust detection and quantification of multiple target nucleic acids in a single sample by using complementary probes labeled with different fluorescent labels.
Project Terms: Adopted; analog; animal tissue; Antisense Oligonucleotides; Australia; base; Biological; Blood; Blood specimen; Buffers; Canada; Cell Culture Techniques; Charge; Clinical; clinical application; clinical diagnostics; clinical implementation; Clinical Research; Communicable Diseases; cyanine; Data; Detection; detector; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic tests; Disease; drug candidate; Early Diagnosis; Early treatment; Eukaryota; flexibility; fluorophore; Freezing; Gene Expression; Generations; Genes; Genetic; Genetic Diseases; High Pressure Liquid Chromatography; Human; human disease; Hybrids; Injections; Ion Exchange; Label; Legal patent; Malignant Neoplasms; Measures; Mediating; Medical; method development; Methods; methylphosphonate; microorganism; MicroRNAs; Modality; Modeling; Monitor; novel; Nucleic Acids; Oligonucleotide Probes; Oligonucleotides; Outcome; Patient-Focused Outcomes; Patients; Pharmaceutical Preparations; Phase; phosphoramidite; Plasma; Plasma Cells; Probability; Procedures; Protocols documentation; Reproducibility; Research; research clinical testing; research study; RNA; RNA Interference; Sampling; scale up; Scheme; Serum; Side; Small Interfering RNA; Specificity; Standardization; Survival Rate; Testing; Therapeutic; Time; Tissue Sample; Tissues; tool; Untranslated RNA; Urine; Vertebral column; Whole Bl
