Boston University

Studying DNA-protein interactions using nanopore force spectroscopy

Exo I is a 3′ - 5′ progressive exonuclease that degrades single-stranded DNA (ssDNA) in the presence of magnesium. Previous studies suggest a model where digestion is initiated by Exo I binding to the 3’ terminus followed by a magnesium dependent hydrolysis of nucleotides from the 3’ end. We use a single alpha-Hemolysin protein nanopore to detect and characterize the interactions between Exo I and ssDNA.

Our method consists of two types of experiments:

1. First, the protein nanopore is used to distinguish and measure the fraction of DNA-protein complexes / free DNA, in solution. Translocations of the lone DNA molecules through the pore cause brief ion current blockades, while the DNA-protein complexes produce much longer blockade events (see Figure 1), which can be readily distinguished and quantified.

2. Second, we use nanopore force spectroscopy to probe dissociation of ssDNA-Exo I complexes captured by the nanopore. Electrical force applied to individual ssDNA-Exonuclease I complexes pulls the two molecules apart, while the ion current probes the dissociation rate of the complex, as shown in the movie (Figure 2). Nanopore Force Spectroscopy reveals energy barriers affecting molecular dissociation, and can be used to determine the association and dissociation constants of the system.

Our method is general and can be applied to study many other nucleic acid-protein complexes.

References:

  1. Hornblower, B., A. Coombs, R.D. Whitaker, A. Kolomeisky, A. Meller and M. Akeson. 2007. Rapid Single-Molecule Kinetic Analysis of DNA-Protein Complexes. submitted
  2. Mathé, J., A. Arinstein, Y. Rabin and A. Meller. 2006. Equilibrium and irreversible unzipping of DNA in a nanopore. Europhys. Lett. 73(1), 128-134
Figure 1
Figure 2