Secondary Structure Detection Through Direct Nanopore RNA Sequencing

Techniques that can directly sequence RNAs and determine secondary structures are essential to establish how an RNA molecule folds and how folding affects its function. We describe the development of a direct RNA nanopore sequencing technique using an engineeredBombyx moriretroelement reverse transcriptase (RT) to thread RNA through theMycobacterium smegmatisporin A (MspA) nanopore in single-nucleotide steps. First, we establish the map that correlates RNA sequence and MspA ion current. We find that during sequencing the RT can sense the strength of RNA secondary structures 11-12 nt downstream of its front boundary, modifying its stepping dynamics accordingly. Using this feature, we achieve simultaneous RNA nanopore sequencing and structure detection, without the need of prior conversion to complementary DNA (cDNA) or RNA modifications. The sequence-dependent ion currents open the way to utilize the MspA nanopore to investigate the single-molecule activity of other processive RNA translocases such as the ribosome.