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Nanopore technology proves useful for analyzing DNA-protein complexes one by one

Nanopore technology proves useful for analyzing DNA-protein complexes one by one
Nanopore technology proves useful for analyzing DNA-protein complexes one by one
Wednesday, April 4, 2007

Research performed by graduate student Breton Hornblower in the UCSC laboratory of Mark Akeson, adjunct associate professor of biomolecular engineering, has yielded a new method for analyzing the strength and kinetics of DNA-protein complexes. The method, developed in collaboration with Amit Meller at Boston University, was published online in Nature Methods on March 4.

Hornblower, who has since moved to industry, aimed to quantify the strength and specificity of DNA-protein interactions using a single-molecule method. This is an alternative to commonly used bulk biochemical and biophysical methods and to other more laborious single-molecule techniques such as optical tweezers.

Measuring single molecules can reveal information, such as bond strength and the presence of short-lived intermediate states, that can be masked by the effect of averaging in experiments using bulk methods.

But to generate meaningful results, a single-molecule analysis method requires the ability to rapidly analyze hundreds of copies of the same molecule. Thus, speed is of the essence.

Enter the alpha'hemolysin nanopore, a method pioneered by the Akeson laboratory at UCSC. The nanopores are narrow ion channels that can be used to analyze single RNA and DNA molecules. Nanopore technology makes it possible to measure DNA structure and dynamics with precision at the angstrom level'on the order of one ten-millionths of a millimeter.

The Akeson laboratory fixes alpha-hemolysin nanopores in a two-ply layer of lipids. They then apply a voltage across the nanopore, with a positive charge on one side and a negative charge on the other side. This causes negatively-charged chloride ions and positively-charged potassium ions to move through the pore in opposite directions, creating a steady current.

The applied voltage also sucks the DNA molecules through the nanopore, which is narrow enough to admit only one molecule at a time in single-file order. When the DNA molecule slides through the nanopore channel, it blocks the current temporarily. Measurements of the current as the DNA molecule slides through yield clues to the molecule's structure.

Hornblower applied this technique, known as nanopore force spectroscopy (NFS), to study biologically relevant interactions between DNA and a protein, the bacterial enzyme Exonuclease I. He did so by placing a solution containing single strands of DNA and the enzyme on one side of the lipid layer.

If one end of a DNA molecule happens to bind to the enzyme molecule, the resulting complex is too large to pass through the nanopore, so the DNA hangs in the passage. This blocks the current for an extended period, until the DNA strand finally detaches from the enzyme and passes through. The time it takes for the bond to break so that the DNA can pass through gives information about the kinetic interaction between the Exonuclease I and the DNA

'The nanopore method described in Breton' paper was able to reveal details of the rate of binding interactions between Exonuclease I and DNA that would be difficult to detect by other methods. So we are excited about the power this method shows for analyzing interactions between DNA, RNA, and enzymes,' Akeson said.

They chose Exonuclease I for this study to further a broader goal of their project: to find a way to control the timing of DNA translocation across the lipid membrane through the nanopore, so that a detector in the pore can read the sequence as the DNA moves through.

'In our view, there can be no better way to do this than using exonucleases or polymerases that cut or synthesize DNA strands one base at a time,' Akeson explained. 'Breton's paper in Nature Methods is the first report to show that one of these enzymes'Exonuclease I'can be coupled to a nanopore.'