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

Abstract

The elegant architecture of the channel of bacteriophage phi29 DNA packaging motor has inspired the development of biomimetics for biophysical and nanobiomedical applications. The reengineered channel inserted into a lipid membrane exhibits robust electrophysiological properties ideal for precise sensing and fingerprinting of dsDNA at the single-molecule level. Herein, we used single channel conduction assays to quantitatively evaluate the translocation dynamics of dsDNA as a function of the length and conformation of dsDNA. We extracted the speed of dsDNA translocation from the dwell time distribution and estimated the various forces involved in the translocation process. A ~35-fold slower speed of translocation per base pair was observed for long dsDNA, a significant contrast to the speed of dsDNA crossing synthetic pores. It was found that the channel could translocate both dsDNA with ~32% of channel current blockage and ~64% for tetra-stranded DNA (two parallel dsDNA). The calculation of both cross-sectional areas of the dsDNA and tetra-stranded DNA suggested that the blockage was purely proportional to the physical space of the channel lumen and the size of the DNA substrate. Folded dsDNA configuration was clearly reflected in their characteristic current signatures. The finding of translocation of tetra-stranded DNA with 64% blockage is in consent with the recently elucidated mechanism of viral DNA packaging via a revolution mode that requires a channel larger than the dsDNA diameter of 2 nm to provide room for viral DNA revolving without rotation. The understanding of the dynamics of dsDNA translocation in the phi29 system will enable us to design more sophisticated single pore DNA translocation devices for future applications in nanotechnology and personal medicine.

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

“…Previous studies reported that the MspA nanopore may provide improved spatial resolution, which has a shorter narrow region than the α -HL channel 6 . The phi29 connector is a rare conic shaped biological nanopre in previous experimental studies, which has been used successfully for single molecule detection and produces higher current signal amplitude in comparing to other biological pores 7,53 . Therefore, the bigger the difference between R and r is, the larger the ionic current and current amplitude are.…”

Fingerprinting of Peptides with a Large Channel of Bacteriophage Phi29 DNA Packaging Motor

“…This is supported by the profile of unfolded TAT peptide (Figure d) compared to folded peptides (Figure a). The blockage of 32% has been reported previously to represent the translocation of dsDNA, which also displays a diameter of 2 nm. From Figure , it is also evident that each of the four blockage profiles show a unique blockage peak with large but different blockage percentages that is representative of the characteristic fingerprint or signature of each peptide.…”

Nanopore Sensing

“…50, 130, 131 The crystal structure of the phi29 motor has been reported previously, 115 and a variety of engineering modifications can be performed to tune the functionality of the pore for specific applications. For example, an engineered phi29 motor pore with reduced channel size has been used to discriminate between translocation of ssDNA and RNA.…”

“…Reported applications of these protein pores include detection of metal ions, 26 small molecules, 27, 28 nucleotides, 29–36 and proteins, 37–48 as well as differentiation between classes of nucleotide conformations. 49, 50 Proof-of-principle studies of DNA sequencing have been realized by a combination of engineered biological nanopores with polymer-ase-based positional control. 51 More recently, nanopore-based sequencing-by-synthesis (SBS) has been developed.…”