Voltage controlled shutter regulates channel size and motion direction of protein aperture as durable nano-electric rectifier-----An opinion in biomimetic nanoaperture

Abstract

In optical devices such as camera or microscope, an aperture is used to regulate light intensity for imaging. Here we report the discovery and construction of a durable bio-aperture at nanometerscale that can regulate current at the pico-ampere scale. The nano-aperture is made of 12 identical protein subunits that form a 3.6-nm channel with a shutter and "one-way traffic" property. This shutter responds to electrical potential differences across the aperture and can be turned off for double stranded DNA translocation. This voltage enables directional control, and three-step regulation for opening and closing. The nano-aperture was constructed in vitro and purified into homogeneity. The aperture was stable at pH2-12, and a temperature of -85C-60C. When an electrical potential was held, three reproducible discrete steps of current flowing through the channel were recorded. Each step reduced 32% of the channel dimension evident by the reduction of the measured current flowing through the aperture. The current change is due to the change of the resistance of aperture size. The transition between these three distinct steps and the direction of the current was controlled via the polarity of the voltage applied across the aperture. When the C-terminal of the aperture was fused to an antigen, the antibody and antigen interaction resulted in a 32% reduction of the channel size. This phenomenon was used for disease diagnosis since the incubation of the antigen-nano-aperture with a specific cancer antibody resulted in a change of 32% of current. The purified truncated cone-shape aperture automatically self-assembled efficiently into a sheet of the tetragonal array via head-to-tail self-interaction. The nano-aperture discovery with a controllable shutter, discrete-step current regulation, formation of tetragonal sheet, and one-way current traffic provides a nanoscale electrical circuit rectifier for nanodevices and disease diagnosis.

Keywords: Nano-aperture; Nano-rectifier; Nanobiotechnology; Nanoelectromechanical system; Nanoelectronics; Nanomotor.

Fig. 1.. Structure of the camera-like nano-aperture.

(A-D) Structure of a camera aperture with stepwise-closing. (E) Crystal structure of the connector of phi29 DNA packaging motor showing the tilting of the 12 protein-subunit with 30° angle between two adjacent connector subunits. (F) Top: Illustration of one 360°-circle divided into 12 sections, showing the shift of 30° angle between two adjacent sections. Bottom: Illustration of one 360°-helical turn of dsDNA divided into 12 steps, showing the shift of 30° angle between two sequential steps.

Fig. 2.. Images of the pacified nano-aperture.

(A-C) TEM images of the fabricated purified nano-aperture, self-assembled into a tetragonal array. The scale bar is 25 nm. (D) Side view of the nano-aperture crystal structure. (E-F) Computationally constructed structure displaying the array structure assembled by alternative up and down arrangement, showing dimensions and polarity.

Fig. 3.. One-way traffic of a nano-aperture.

(A-B) “One-way” DNA translocation of the connector of phi29 DNA packaging motor under a ramping potential from −100mV to +100mV (data from⁴²⁾. The change of the connector orientation leads to the appearance of current blockage spikes when dsDNA was placed at both trans and cis sides of the chamber (data from⁴²⁾. (C) No DNA in either chamber as negative control (data from⁴²⁾. (D) Alteration of voltage polarity to demonstrate that DNA can only be translocated from one side at one kind of polarity, when DNA was placed in both trans and cis chambers(data from⁴²⁾. (E) Illustration showing the flexible inner channel loops (green color) of the phi29 connector. (F) Two-way traffic property of peptide through the internal loop-deleted Phi29 connector. Figures were adapted with permission from Copyright 2013 American Chemical Society.

Fig. 4.. Three-step gating of connector nano-aperture of phage DNA packaging motors.

The observed dip in (A) into the 2^nd step of the phi29 conformation while still in the 1^st step indicates that the gating conformational changes are reversible and there are varying degrees of stability within each of the conformational states.

Fig. 5.. Real-time sensing of EpCAM antibody (Ab) interactions with EpCAM antigen (Ag) using engineered phi29 connector nano-aperture, demonstrating the C-terminal is the shutter triggering the step-wise conformational change.

(A) Schematic diagram showing the placement of the colon cancer Ag into the C-terminal of the phi29 connector for real-time detection of the Ag with its corresponding Ab. (B) Histogram of current blockage events caused by the addition of diluted EpCAM Ab. (C) Histogram plotting of current transition events of specific (green) and nonspecific (red) binding to the shutter at the C–terminal caused by Ab/Ag interactions.