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Oxford Team Demonstrates Passage of Long RNA Heteropolymers through Protein Nanopore

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Researchers at the University of Oxford working with an alpha-hemolysin protein-based nanopore sequencing strategy have shown that it is possible to pass long RNA heteropolymers through alpha-hemolysin pores using an electric current.

The group, led by Hagan Bayley, who is also a co-founder of Oxford Nanopore Technologies, reported its results online last month in the journal Nano Letters, showing that longer than expected, and almost complete ionic current blockages, were clear evidence of the passage of these long RNA molecules through the pore.

The study's first author, James Cracknell, a postdoctoral fellow in Bayley's University of Oxford lab, said that researchers within the Bayley lab and elsewhere in protein nanopore research, have previously focused their work on shorter DNA or RNA molecules or have used homopolymers because they are less likely to form complex structures, and thus are more easily threaded through a nanopore.

In the new study the group wanted to show that long molecules with the potential to tangle up could also be translocated through alpha-hemolysin pores, Cracknell told In Sequence this week.

"In previous years and publications, effort has been taken to avoid complications from the complex structure of polymers, either by using homopolymers, which have limited potential for base pairing, or by using very short strands— typically under 100 bases. But in this study, we wanted to look at whether it was possible to get much longer, much more structurally complex molecules to go through the pore, and we showed that it is possible."

Using RNA served two purposes in the study, Cracknell said. First, there is a lot of interest in single-molecule pore-based sequencing of RNA itself, including among the Oxford lab team, which last year showed, using short homopolymers, that it is possible to use alpha-hemolysin nanopores to recognize individual RNA bases (IS 10/16/2012).

Additionally, although Cracknell said his team is interested in the translocation of long heteropolymers in general, including DNA, RNA — which can become even more solidly tangled than analogous DNA molecules — provided the best experimental challenge for the group.

"We chose RNA as it was actually more difficult. If we could do RNA, that would be good evidence that we could do this for DNA as well," he said.

In the study, Cracknell and his colleagues measured current changes as RNA heteropolymers between 91 bases and 6,083 bases were subjected to voltage to drive them through an alpha-hemolysin nanopore.

Blocks in the current, an indication that a molecule is inside the pore, were measured for varying strand lengths and under different voltage conditions.

These current blockades, according to the authors, decreased in duration with higher voltage and increased in duration in a linear fashion for longer and longer molecules — both evidence that the blocks represented RNA translocation through the protein pore.

The group further proved that the current blockages were due to translocation by attaching the protein streptavidin, which cannot pass through an alpha-hemolysin pore, to the RNA heteropolymers. With the streptavidin-bound RNA, the researchers observed that half of the blockage events were permanent blocks in current, requiring the group to reverse the voltage polarity to back the molecules out of the pore. Without streptavidin present, only 10 percent of the blockages were similarly permanent.

This difference provided "strong evidence," according to the researchers, that the observed transient blockages were indeed caused by the translocation of the long RNA molecules through the pore.

Interestingly, the length of the current blockages the group saw as the long RNA molecules translocated — between about one and 10 milliseconds per base — were "orders of magnitude" greater than what researchers have observed with short pieces of DNA — one-to-10 microseconds per base, Cracknell said.

He noted the group can't be sure what causes the long molecules to translocate at a slower rate, but that it is likely due to complex secondary structures that form in heteropolymer RNA lengths, including local and long-range base pairing, as well as what the group called in the paper a "blob" structure, where the molecule forms a tangle, or "ball-of-string" structure that becomes somehow energetically stable.

What we imagine right now is that the end of the RNA is threaded into the pore, and then once we see the current blockage event, this blob [of RNA] is being unwound by the force of voltage we apply. "It probably doesn't unwind all in one step; there is probably partial translocation and then a little more unwinds, and then more translocation, and then more unwinds … and then when it has fully translocated, the current goes back to normal," Cracknell said.

According to Cracknell, the group's results have some interesting potential implications for alpha-hemolysin pore sequencing. For example, he said, the speed at which short polymers translocate is too fast to be able to measure the signal of individual bases passing through the pore over background noise.

The longer passage times the group measured using long RNA could theoretically, he said, be long enough for individual base-pairs to be measured.

That, however, depends on whether the RNA molecules are moving smoothly through the pore at this slower speed, or are slipping through in quick spurts punctuated by pauses— something the team does not yet know, according to Cracknell.

"That would affect whether this is a feasible system to measure individual bases," he said.

Another surprise in the results, Cracknell said, was that the translocation of the long RNA heteropolymers caused a more-complete current blockage than what researchers have seen in earlier experiments with short molecules.

This could also potentially influence whether sequencing is possible, according to Cracknell. "If you are measuring bases by how much current is able to get past them, that's another level of complexity," he said.

According to Cracknell, the main thrust of research toward using alpha-hemolysin pores for sequencing is now in trying to precisely control the passage of molecules through the pore.

Researchers from last year's Oxford study using short RNA homopolymers told In Sequence at the time that they were looking at enzyme-based options for ratcheting molecules through the pore in a controlled fashion, potentially similar to the approach that the University of Washington's Jens Gundlach and University of California, Santa Cruz, researcher Mark Akeson have described for DNA sequencing with the MspA pore protein (IS 3/27/2012).

Cracknell said he is not involved with Oxford Nanopore so couldn't comment on the implications of the research for the firm's commercial plans nanopore sequencing.

Last year, Oxford Nanopore announced that it planned to introduce two commercial nanopore sequencing instruments, a scalable GridIon platform and a USB-stick sized MinIon sequencing system during the year (IS 2/21/2012).

The company also did not provide a public update on the status of its technology or launch dates for the instruments at this year's Advances in Genome Biology and Technology meeting (IS 2/26/2013).

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