A Novel Method for the Capture-based Purification of Whole Viral Native RNA Genomes

Abstract

Current technologies for targeted characterization and manipulation of viral RNA either involve amplification or ultracentrifugation with isopycnic gradients of viral particles to decrease host RNA background. The former strategy is non-compatible for characterizing properties innate to RNA strands such as secondary structure, RNA-RNA interactions, and also for nanopore direct RNA sequencing involving the sequencing of native RNA strands. The latter strategy, ultracentrifugation, causes loss in genomic information due to its inability to retrieve unassembled viral RNA. We developed a novel nucleic acid manipulation technique involving the capture of whole viral native RNA genomes for downstream RNA assays using hybridization baits in solution to circumvent these problems. This technique involves hybridization of biotinylated baits at 500 nucleotides (nt) intervals, stringent washes and release of free native RNA strands using DNase I treatment, with a turnaround time of about 6 h 15 min. Proof of concept was primarily done using RT-qPCR with dengue virus infected Huh-7 cells. We report that this protocol was able to purify viral RNA (561-791 fold). We also describe a successful application of our capture-based purification method to direct RNA sequencing, with a 77.47% of reads mapping to the target viral genome. We observed a reduction in human host RNA background by 1580 fold, a 99.91% recovery of viral genome with at least 15x coverage, and a mean coverage across the genome of 120x. This report is, to the best of our knowledge, the first description of a capture-based purification method for whole viral RNA genomes. The fundamental advantages of using our capture-based purification method makes it a superior alternative to conventional viral purification methods and would potentially pave a new path for the direct characterization and sequencing of native RNA molecules.