Redox Engineering of Cytochrome c using DNA Nanostructure-Based Charged Encapsulation and Spatial Control

Abstract

Three-dimensional (3D) DNA nanostructures facilitate the directed self-assembly of various objects with designed patterns with nanometer scale addressability. Here, we report the enhancement of cytochrome c (cyt c) redox activity by using a designed 3D DNA nanostructure attached to a gold electrode to spatially control the position of cyt c within the tetrahedral framework. Charged encapsulation and spatial control result in the significantly increased redox potential and enhanced electron transfer of this redox protein when compared to cyt c directly adsorbed on the gold surface. Two different protein attachment sites on one double stranded edge of a DNA tetrahedron were used to position cyt c inside and outside of the cage. Cyt c at both binding sites show similar redox potential shift and only slight difference in the electron transfer rate, both orders of magnitude faster than the cases when the protein was directly deposited on the gold electrode, likely due to an effective electron transfer pathway provided by the stabilization effect of the protein created by the DNA framework. This study shows great potential of using structural DNA nanotechnology for spatial control of protein positioning on electrode, which opens new routes to engineer redox proteins and interface microelectronic devices with biological function.

Keywords: DNA tetrahedron; cytochrome c; framework nucleic acids; protein engineering.

Figure 1.

DNA tetrahedron is formed by self-assembly of four oligonucleotides. (a) Three strands are thiol-modified to anchor the DNA tetrahedron onto the gold electrode, and the fourth strand is site specific, covalently linked to cyt c, to form the DNA tetrahedrons named T₁ and T₂. (b) SMCC was used as the bispecific linker between the DNA and the protein, with the distances marked between the terminal end of the DNA and the heme group of cyt c. (c) A simple scheme illustrating the positioning of the protein molecule inside or outside the edge of the DNA tetrahedron. The arrows point to the protein attachment sites at the 5′ end of one of the nick points on one of the duplex edge. (d) Cryo-EM 3D reconstruction and comparison between representative class averages (C) and corresponding 3D map projections (P) of DNA tetrahedron T₀. Twenty angstrom scale bar is shown in panel d. (e) Left: Native PAGE (6%) characterization of the assembled DNA tetrahedron with cyt c protein inside and outside. Lane 1: T₀; Lane 2: T₁; Lane 3: T₂. Right: Fluorescence gel image of an Alexa 555-labeled cyt c-DNA tetrahedron structure. Lane 2: Alexa 555-labeled T₁; Lane 3: Alexa 555-labeled T₂. Lane M: DNA ladder. (f) Left: Cryo-EM SPA reconstruction of T₀ and the corresponding cutaway views, applied with a Gaussian filter low passed to 38 Å. Right: Cryo-EM subtomogram averaging of cyt c occupied T₁ and the corresponding cutaway views at spatial resolution of 38 Å. Twenty angstrom scale bar is shown in panel f.

Figure 2.

Cyclic voltammetry scans of cyt c accommodated by the DNA tetrahedron structure on the gold electrode. (a) T₁. (b) T₂. All CV scans were taken at a scan rate of 50 mV/s using reference electrode Ag/AgCl (3 M).

Figure 3.

(a) Comparison of the UV–vis spectra of the wild-type cyt c (black) and DNA–cyt c conjugate (red) of same concentration in HEPES buffer (2 mM; pH 7.4; 25 °C). Left panel: Both samples were dissolved in HEPES buffer without any treatment. Right panel: Reduced cyt c was prepared from the wild-type cyt c, treated with TCEP overnight with the TCEP subsequently removed the following day. The same DNA–cyt c conjugate spectrum in the left panel is shown for comparison. (b) Comparison of circular dichroism spectra of the wild-type cyt c (black) and DNA–cyt c conjugate (red) in HEPES buffer. Left panel: Far-UV (250–180 nm) region. Right panel: Soret (450–350 nm) region. The CD spectra were scanned at 30 nm/min and averaged from 4 scans.

Figure 4.

Alternating current voltammetry scans and plot of I_p/I_b vs log(frequency). (a) T₁. (b) T₂. The ACV data were collected at 5 Hz and baseline-subtracted. In the I_p/I_b plot, the dots are the experimental data; the solid curves are theoretical fitting following the method reported previously.