α-Hemolysin nanopore studies reveal strong interactions between biogenic polyamines and DNA hairpins

Abstract

The α-hemolysin (α-HL) nanopore analyzes DNA as it is electrophoretically driven through the pore. The respective current vs. time (i-t) traces depends on the DNA sequence, its secondary structures, or on the physical conditions of the analysis. The current study describes analysis of a DNA hairpin with a 5'-extension with the α-HL nanopore in the presence of the polyamines spermine (Spm), spermidine (Spd), and putrescine (Put). These studies identified a new i-t trace characteristic of the DNA-polyamine complex. Voltage-dependent studies determined that the hairpin-Spm complex formed with excess Spm was not unzipped and translocated through the pore even when the voltage was increased to 180 mV. The DNA hairpin sample was titrated with Spm, Spd, or Put that showed a dose-dependent response in the characteristic event patterns for hairpins bound to Spm or Spd, but not for Put. Plots of the event types vs. count were used to calculate binding constants for the Spm or Spd hairpin interactions under these conditions. The titration also demonstrated that the event rate decreased ~10-fold when the Spm or Spd concentration was increased from 0 to 4 mM. These observations impose practical limitations on the ability to use Spm or Spd for DNA studies with the α-HL nanopore.

The applications of nanopores in studies of proteins

“…Nanopores can have different measurement capacities based on the materials used and can be divided into two major types: biological nanopores and solid-state nanopores. Biological nanopores are made of proteins with funnel-shaped structures, such as a-hemolysin [25][26][27][28][29][30][31][32], Msp A [18] and Phi29 [33]. Solid-state nanopores are made of nonbiological materials [34,35], such as silicon nitride [36,37], quartz [38], and graphene [39][40][41][42][43].…”