Profiling miRNAs using a nanopore device and two nucleobase-selective tags

PROJECT SUMMARY Cost effectiveand accurate sequencing of RNAcomposed of both canonical and modified basesof any lengthwithout conversion to cDNAand without amplification are the objectives of this projectand the ultimate goal is to sequence the transcriptomeand determine in a time sensitive manner relative distribution of its componentsSuch accomplishment will directly impact preventiondiagnosisand cure of diseaseand materialize the promise of personalized medicineCurrent methodssuch as Illumina s RNA Seqand the single molecule approaches of Pacific Biosciences and of Oxford Nanopore TechnologiesONTstill lag behind in many of the critical attributes mentioned aboveNanopore based sequencing has made amazing strides the last few yearsand its ability to provide results in the field can t be understatedThe unresolved issue with nanopore based sequencing is the observation that ion current vstime recording does not refer to a single nucleobasebut to a short sequence oftobasesThe problempartially resolved with the use of sophisticated algorithms and match to a referenceappears intractable for de novo sequencing of RNA known to include numerous post transcriptional base modificationsAs an examplefor a nanopore that senses a sequence ofbases and a specific RNA with a total ofdifferent nucleobasescanonical andmodifiedsequencing will requiredistinct signalsto be discriminated from within an ion current range oftopA with a standard deviation ofpAan impossible computational taskOn the contraryif the nanopore could senselet us saytwo bases at a time and yield distinct ion current for each doubletthere will be onlydifferent recordings to distinguish froma computationally simple taskPublished results in collaboration with Northeastern University in Boston and University of Utah in Salt Lake City clearly indicate that oligodeoxynucleotides tagged with a pyrimidine specific labelOsmium tetroxidebipyrimidineOsBpyield enzyme freeslow readable translocation via solid state pore as well as via a protein poreHemolysinDistinct ion current levels were observed for intactdT OsBpand dC OsBpbasessuggesting that a single tag can yield sequencing information on deoxypurinedTand dCPreliminary results at Yenosunder a Phase I RSBIRgrantusing synthetic RNAs and the MinION device from ONT extend the above findings to RNAand suggest a two nucleobase sensing regime for RNA OsBpHere the proposition is made that the presence of a secondguanosineselectivelabel will facilitate identification of all four canonical basesandlikelyextend identification to many modified RNA basesMost importantly the intrinsic selectivity of a label for one base over anotherwill provide a handle for additional discrimination among the modified basesLabeling is inexpensivetakes couple of hours at room temperaturerequires a simple mixing of sample with the labelfollowed by aminute validated purification stepand could be accomplished using a kitIn this Phase I proposal we aim to demonstrateNearlabelingtrue positivespractically negligible internucleotide bond cleavageandnegligible false positives for RNA that is osmylated at the pyrimidinesas well as platinated atguanosineRNA OsBp PtReadable translocation via the MinIONor an alternative nanopore platformwith a two basesensing regime for RNA OsBp PtandProfiling sequencingtonucleotide long synthetic RNAs in a mixtureas a proof of principlefor a miRNA profile assayand the extension of the technology to medium size RNAsSuccess in these efforts may lead to the development of an inexpensivenon invasive medical diagnostic test for a broad range of disease and well being conditionsi ea miRNA profile assay from biological fluidsA nanopore based miRNA panel assay will also pave the way for sequencing the transcriptomeincluding identification of post transcriptionally modified baseswithout the need for sample amplificationsample library preparationor cDNA synthesis PUBLIC HEALTH RELEVANCEAdvances in personalized medicine for diagnosis and treatment of disease require sequencing the RNA transcriptome with technologies that are cheapfastand accurateCurrent technologies have made big stridesbut still lag behind in accuracy and or expensetake weeks from time of taking sample to medical resultsand leave post transcriptionally modified RNA bases unidentifiedNanoporebased platforms devices that exhibit a two base sensinglike the one addressed in this proposalwill improve sequencing of the transcriptome by simplifying sample processingby identifying all the nucleobasescanonical or modifiedby including all RNAsi eshortlong and ones with no poly Atailand by improving accuracy from currentto over