Despite large investments in nucleic acid technology, the ability to sequence large numbers of full-lengthindividual RNAs, from complex samples, with highest accuracy, has remained out of reach. Pheno introducesan advance for large scale Next Generation DNA Sequencing (NGS) sequencing of effectively unlimited numbersof individual RNA molecules from heterogeneous mixtures. We seek to prove that these methods can beimplemented with the scale and precision to justify translation into commercially viable products and services.The aims of this proposal address two key steps that determine scale and accuracy at which the technology canbe applied, exploiting recent discoveries in nucleic acid enzymology. Aim #1 focuses on reverse transcription,prompted by creation, with in vitro directed evolution, of a highly accurate, proof-reading reverse transcriptase,with high processivity and devoid of integral RNase H activity and other sources of RT artefacts. This advanceover even the best retroviral and enzymes derived from retrotransposons or group-ii introns promises accuratereplication of even the longest RNA viral genomes. Conventional retroviral enzymes, for example, even modifiedby site directed mutagenesis, are prone to at least 11 transcriptional artefacts in addition to simple base-readingerrors. Aim #2 exploits a recently discovered class of primer-polymerases used in DNA repair. A key step in oursequencing chemistry exploits topological advantages of homo-concatamers of tagged cDNAs produced byRolling Circle Amplification (RCA) of circularized single-stranded templates. The lowest scale at which thetechnology can be applied is limited by conventional RCA protocols that depend on exogenous random DNAprimers. and generate artifactual sequences with samples of small size. New protocols use a PrimPolpolymerase to synthesize RNA primers directly from the template, preventing de novo artefacts andsimultaneously improving amplification by ~ 5 orders of magnitude. This presents the exciting possibility ofapplying the technology to samples below the scale of a single-cell transcriptome. Collectively these studies willseek to set new industry standards for RNA sequencing. This could help accelerate a wide range of precisionmedicine, viz. precision cancer diagnostics, immunotherapy; therapeutic gene editing; new drug discovery andvalidation. The technology could provide transformational advances in battling infectious diseases, includingHIV/AIDS and SARS-CoV-2-mediated COVID 19.
Public Health Relevance Statement: Narrative
In the past 20 years the technology for extremely high throughput DNA sequencing has not taken into account
that for human genes, the dictum that applies is one-gene:multiple-proteins. Phaeno NGS based RNA
sequencing can reveal the full diversity of individual RNA molecules in a complex sample, at almost unlimited
scale. This technology provides the foundation to entirely new approaches to gene-based diseases such as
Cancer or Alzheimer's diseases - and can provide tremendous leverage in the battle against RNA viruses such
as the novel SARS-CoV-2 coronavirus.
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