The University of Oxford laboratory behind the alpha-hemolysin protein-based nanopore DNA sequencing strategy being developed by Oxford Nanopore Technologies has shown that it is possible to use similar nanopores for recognizing RNA bases.
As they reported in the journal Nano Letters, Hagan Bayley and Mariam Ayub, a postdoctoral fellow in Bayley's lab, compared the signals associated with immobilized RNA oligonucleotides in wild type and modified alpha-hemolysin pores — a strategy similar to that used to discriminate between anchored DNA bases during preliminary studies that provided the foundation for alpha-hemolysin nanopore sequencing of DNA.
The proof-of-principle study demonstrated that modified alpha-hemolysin proteins with slightly larger than wild-type pore diameters produced distinguishable signals for all four RNA bases, as well as three modified bases.
"Nanopore sequencing for RNA is still quite a new subject — most people have focused on DNA," Ayub says. "The idea behind our paper was to really just put some light on whether we can actually sequence RNA in the same way as DNA."
The technique that they used for recognizing each RNA base in the pore was quite similar to that described for DNA sequencing with nanopores, Ayub says, though RNA is somewhat less stable than DNA and is prone to forming secondary structures.
"If you have a single strand of RNA, it has hairpins and loops in its structure, so it's not linear," she says. "When it passes through the pore, you have to unfold it."
That was not an issue for the stretches of single-stranded RNA that researchers were dealing with in the current study, which were relatively short and immobile. But it is a consideration when dealing with longer pieces of RNA.
Fortunately, Ayub adds, researchers think that the same sorts of ratcheting methods that will be needed to translocate RNA strands through the nanopore at speeds suitable for sequencing should also iron out such kinks in the molecule.
To that end, she is currently looking at enzyme-based options for processively sequencing RNA molecules as they move through the alpha-hemolysin pore.