Nanopore Recordings to Quantify Activity-Related Properties of Proteins - Nanopores: Sensing and Fundamental Biological Interactions - page 216

the ligand reduced the frequency of the current blockages or resulted in long current
blockages that indicated the capture of a protein-ligand complex (Fig. 9.6 ). Thus,
similar to the approach mentioned above, k off values could be estimated from the
duration of the long current blockades by ( 9.7 )[ 24 , 33 ]. Kasianowicz et al . first
demonstrated this technique using wild type

-hemolysin pores and biotinylated,
single-stranded DNA in order to detect the binding of the protein NeutrAvidin to
biotinylated DNA [ 24 ]. The authors used short biotinylated DNA strands (10 mer)
to demonstrate that protein binding to the ligand-modified polymers reduced the
frequency of the current blockages. In addition, the authors observed that protein
binding to long biotinylated DNA (50 mer) strands resulted in permanent current
blockages (permanent because NeutrAvidin binding to biotin is essentially irrevers-
ible K d ~10 15 M) [ 26 ].

a

Fig. 9.6 Detecting the binding of proteins to ligand molecules that were attached to polymers
dissolved freely in the bulk solution. Unbound polymers passed through the pore and resulted in
transient current fluctuations. Depending on the length of the polymer, binding of the protein to the
ligand on short polymers (10 mer DNA) reduced the frequency of the current blockades, while
binding of protein to the ligand on long polymers (50 mer DNA) resulted in current blockades with
a duration proportional to k off . Adopted from Kasianowicz et al . [ 24 ]