Direct tracking of reverse-transcriptase speed and template sensitivity: implications for sequencing and analysis of long RNA molecules

We combined electrophoresis and nanopore sequencing to analyze MarathonRT velocity over the heterogeneous sequences and structures of HOTAIR RNA template and reveal that the local sequences and structures of the template have negligible effect to MarathonRT, and MarathonRT can copy the long RNA in a single turnover, which leads to unusually synchronized primer extension at a constant speed of 25 nt/sec. We further demonstrate that ultra-stable RNA structure insertions do not obstruct MarathonRT, suggesting that MarathonRT can immediately disrupt any RNA structures within the template. Overall design: MarathonRT velocity was determined using the cDNA samples generated at different reaction times (from 5 to 70 sec with 5 sec increment). The RT stops were confirmed using two replicates.