Jan 22, 2010
Nanopores could unravel RNA
A team at the Delft University of Technology in the Netherlands has succeeded in capturing single molecules of RNA inside a nanopore for the first time and measuring the forces on them. The technique could be used to determine the shape of RNA molecules – something that is difficult to do – and potentially reveal their complicated structures.
The technique has been demonstrated for DNA before, but never for RNA, says team member Michiel van den Hout of the Kavli Institute at Delft. RNA is an important and very interesting biological molecule because it can fold into complex shapes. These determine how the molecules interact with other molecules in cells and thus how RNA functions. Moreover, many viruses contain genomes that consist of RNA.
It is still very difficult to determine the shape of RNA molecules, however. With the new technique, developed by Nynke Dekker's team, the molecules could potentially be unfolded by pulling them through nanopores, thus revealing their structure. The method could also allow researchers to detect proteins bound to RNA.
Difficult to handle
"The challenge so far has been that RNA molecules are very small and so difficult to handle," explained van den Hout. "Our measurements demonstrate a new technique that allows us 'grab' single RNA molecules, thread them through a narrow hole, pull them back and forth, potentially unfold them and measure forces on them. Such a new tool could greatly benefit our knowledge and understanding of these molecules."
The researchers began by attaching single molecules of double-stranded RNA (dsRNA) to a polystyrene bead in a salt solution. They then optically trapped the bead, that is, they fixed it inside the focus of a high-power laser beam. This technique allowed them to move the bead to any location inside a sample cell.
Next, the team moved the bead near a solid membrane containing a small nanopore. Single molecules could then be captured inside the nanopore because the molecules are negatively charged and are forced inside the nanopore by an applied electrical field. However, because the molecules are also attached to the bead, they remain inside the pore. When the bead is pulled slightly out of the laser focus, an optical restoring force balances the total electrophoretic forces on the bead.
"The bead's displacement from the laser focus is proportional to the net force pulling the molecule inside the nanopore, thereby allowing us to directly determine this force," van den Hout told nanotechweb.org.
The results also show that the net force on dsRNA molecules is similar to that on DNA. This is because dsRNA forms a double helix structure, which is very similar to DNA's.
The team now plans to demonstrate that it can unfold structured RNA molecules using its technique.
The work was published in Nano Letters.
About the author
Belle Dumé is contributing editor at nanotechweb.org