Novel Nanopore-based RNA Sequencing using Nucleobase-specific 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 disease and materialize the promise of personalized medicineCurrent methodssuch as Illuminaandapos s RNA Seqand the single molecule approaches of Pacific Biosciences and of Oxford Nanopore Technologiesstill lag behind in many of the critical attributes mentioned aboveThe 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 ofor more basesThe problempartially resolved with the use of sophisticated algorithms and learning machinesappears intractable for RNA that includes numerous post transcriptional base modificationsAs an illustrationif a nanopore reads a sequence ofbases and the specific RNA to be sequenced has a total ofdifferent nucleobasescanonical andmodifiedthensignals need to be discriminated from within an ion current range oftopA with a standard deviation ofpAthis is an impossible computational taskHoweverif the nanopore could sense one base at a time and yield distinct ion current for each basethere will be onlydifferent recordings to distinguish froma much simpler taskOur own published results indicate that oligodeoxynucleotides conjugated with a pyrimidine specific tagOsmium tetroxideandaposbipyrimidine or OsBpyield enzyme freeslow readable translocation viaHemolysinand distinct ion current levels for intactT OsBpand C OsBpbasessuggesting that a single tag can yield sequencing information on purineTand CThe latter leads to the conjecture that the presence of a secondpurine specificlabel would allow identification of all four canonical basesFurthermore each tag has intrinsic selectivity for one base over anotherand this will provide a handle for additional discrimination among the modified basesIn this phase I proposal we aim to demonstrateinearlabelingtrue positiveswithinternucleotide bond cleavageandfalse positives for RNA OsBpas we have already shown for DNA OsBpiicomparable labeling attributes for a purine specific tagandiiireadable translocation with single pyrimidine base discrimination for RNA OsBpSuccess in these efforts will lead to single base discrimination and sequencing of RNAincluding a number of post transcriptionally modified basesand pave the road for sequencing the transcriptome!PUBLIC HEALTH RELEVANCEAdvances in personalized medicine for diagnosis and treatment of disease require sequencing the RNA transcriptome with technologies that are currently unavailableNanopore systems that exhibit single base discriminationlike the one addressed in this proposalwill allow sequencing the transcriptome in an accuratetimelyand cost effective manner