New-Generation of Fluorescently-Labeled Charge-Neutralized Oligonucleotides as Probes for the Detection and Quantification of MicroRNAs and Oligonucleotide-Based Therapeutics in Biological Samples

ABSTRACTTo datemore thanmicroRNAsmiRshave been identified in humansAberrant miRs expression has been associated with many human diseasesand especially with cancerThereforeearly detection and quantitation of those miRs can have a significant impact on patientsandaposoutcome and survival rateThe 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 samplesNovelty and significance of this proposal is based on the development of ZATA s fluorescent labeled new generation oligonucleotideONbased diagnostic probesZ Probesthat will enable the detection and quantification of several target miRs simultaneously in a single sampleAbility to simultaneously detect several target miRsalong with other advantagessuch as simplicity of procedurehigh sensitivityselectivityreproducibilitymakes our method superior to currently used approachessuch as qPCRThe second and equally important application of Z probes is for detection and quantitation of synthetic ONs in PK PD studies of ONs based therapeuticsthat are widely tested against cancermicroorganism mediatedgenetic and other diseasesZ probes take an advantage on ZATA s novel ONs with controlled charge neutralization of their backbonesOur preliminary data strongly supports the feasibility of Z probes development into diagnostic modalities for research and clinical useAs 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 serumObtained data have demonstrated high feasibility of the proposed probes for diagnosis purposeIn the framework of this applicationwe anticipate achieving several milestones that will validate our Z probes as a diagnostic modality for research and clinical studiesThose milestones areUse of Z probes for quantification of short interference RNAssiRNAantisense ONsand synthetic analogs of miRs in biological samplestissuewhole bloodplasmacell cultureand in urineDemonstration of the sequence specificity and high selectivity of Z probes by testing them against non complimentary and mismatched target nucleic acidsScaling up and optimizing of the method for synthesis of Z probesUse of Z probes for the quantification of endogenous miRs in the cell culturesas well as in human serumblood and tissue samplesThe outcome of this Phase I will be methods and technical protocols for the detection and quantification of antisense oligonucleotidessiRNAsand miRs in biological samples using ZprobesMethod of synthesis of Z probes and composition of matter are claimed in new national stage patent applications in USEUCanadaand Australia NARRATIVEDevelopment of the new generation of fluorescent labeled charge neutralized oligonucleotideONprobes for the detection and quantification of microRNAs and ON based drug in biological samples by using ionexchange HPLC with fluorescent detection is proposedThis 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