Robust properties of membrane-embedded connector channel of bacterial virus phi29 DNA packaging motor

Abstract

SUMMARY Biological systems contain highly-ordered macromolecular structures with diverse functions, inspiring their utilization in nanotechnology. A motor allows linear dsDNA viruses to package their genome into a preformed procapsid. The central component of the motor is the portal connector that acts as a pathway for the translocation of dsDNA. The elegant design of the connector and its channel motivates its application as an artificial nanopore. Herein, we demonstrate the robust characteristics of the connector of the bacteriophage phi29 DNA packaging motor by single pore electrophysiological assays. The conductance of each pore is almost identical and is perfectly linear with respect to the applied voltage. Numerous transient current blockade events induced by dsDNA are consistent with the dimensions of the channel and dsDNA. Furthermore, the connector channel is stable under a wide range of experimental conditions including high salt and pH 2–12. The robust properties of the connector nanopore made it possible to develop a simple reproducible approach for pore quantifications. Such quantifications led to a reliable real time counting of DNA passing through the channel. The fingerprint of DNA translocation in this system has provided a new tool for future biophysical and physicochemical characterizations of DNA transportation, motion, and packaging.

Single pore translocation of folded, double-stranded, and tetra-stranded DNA through channel of bacteriophage phi29 DNA packaging motor

“…1D ) with a channel conductance determined to be 3.1 ± 0.3 nS , calculated using the ratio of the measured current jump (under 1 M NaCl, 5 mM Tris, pH 7.8) resulting from connector insertion to the applied voltage (75 mV). Furthermore, the C-His connectors exhibit a perfectly linear I–V relationship without displaying any voltage gating phenomenon under the reported conditions of ±100 mV, as established previously [17,19-21]. …”

“…The step size of the connector channels was homogenous and the channels exhibited equal conductance under both positive and negative trans-membrane potentials [17,18]. The connectors exhibit a perfectly linear Current-Voltage ( I–V )relationship without displaying any voltage gating phenomenon at ±100 mV [17,19-21]. Furthermore, the nanopore system is stable under a wide range of solution conditions, including pH 2 - 12, and ionic strengths of 0.1 - 3 M NaCl or KCl [19].…”

“…The connectors exhibit a perfectly linear Current-Voltage ( I–V )relationship without displaying any voltage gating phenomenon at ±100 mV [17,19-21]. Furthermore, the nanopore system is stable under a wide range of solution conditions, including pH 2 - 12, and ionic strengths of 0.1 - 3 M NaCl or KCl [19]. …”

Conic shapes have higher sensitivity than cylindrical ones in nanopore DNA sequencing

“…Yet, the system still suffers typically from limitations, including flaw to external vibrations, such as PH, temperature, and mechanical oscillations 2 . The phi29 connector was chemically stable over a range of pH, temperature and ionic strength 11,12 .…”

Oriented single directional insertion of nanochannel of bacteriophage SPP1 DNA packaging motor into lipid bilayer via polar hydrophobicity

“…The phi29 connector forms a dodecameric funnel shaped channel [26; 27] with an internal channel diameter of 6 nm at the wider end and 3.6 nm at the narrowest constriction. We have extensively investigated the membrane-embedded phi29 nanopore system [8; 28] for characterizing nucleic acids [8; 18; 29–34], and for sensing of proteins [2] and chemicals [35]. The phi29 connector exhibits a one-way traffic property for dsDNA translocation from N- to C-terminus [18].…”