Interenzyme Substrate Diffusion for an Enzyme Cascade Organized on Spatially Addressable DNA Nanostructures

Abstract

Spatially addressable DNA nanostructures facilitate the self-assembly of heterogeneous elements with precisely controlled patterns. Here we organized discrete GOx/HRP enzyme pairs on specific DNA origami tiles with controlled inter-enzyme spacing and position. The distance between enzymes was systematically varied from 10 nm to 65 nm and the corresponding activities were evaluated. The study revealed two different distance dependent kinetic processes associated with the assembled enzyme pairs. Strongly enhanced activity was observed for those assemblies in which the enzymes were closely spaced, while the activity dropped dramatically for enzymes as little as 20 nm apart. Increasing the spacing further resulted in a much weaker distance dependence. Combined with diffusion modeling, the results suggest that Brownian diffusion of intermediates in solution governed the variations in activity for more distant enzyme pairs, while dimensionally-limited diffusion of intermediates across connected protein surfaces contributed to the enhancement in activity for closely spaced GOx/HRP assemblies. To further test the role of limited dimensional diffusion along protein surfaces, a noncatalytic protein bridge was inserted between GOx and HRP to connect their hydration shells. This resulted in substantially enhanced activity of the enzyme pair.

A DNA tweezer-actuated enzyme nanoreactor

“…Next, G6pDH was conjugated to a ssDNA (5 0 -TTTTTCCCTCCCTCC-3 0 ) using welldeveloped chemical methods 24 . The complementary anchor strand was displayed from one of the tweezer arms to capture the DNA-modified G6pDH via sequence-specific hybridization.…”

“…N-Succinimidyl 3-(2-pyridyldithio)-propionate was used to crosslink G6pDH with a 5 0 thiol-modified oligonucleotide 24 . Detailed protein-oligo conjugation protocol was shown in Supplementary Methods.…”

“…A three-fold molar excess of oligonucleotide-conjugated G6pDH was added to the pre-annealed tweezer structures and mixed completely. Protein assembly was performed using an annealing protocol in which the temperature was gradually decreased from 37°C to 10°C over a period of 1 h and then held constant at 4°C using an established procedure 24 . Excess G6pDH-WN1 was removed by affinity of the biotin-labeled tweezers using monomeric avidin resin (Pierce).…”

“…Examples of such structures include autonomous walkers 15,16 , nanotweezers [17][18][19][20] and nanocages for controlled encapsulation and payload release 21,22 . New protein-DNA conjugation chemistries make it possible to precisely position proteins and other biomolecules on DNA scaffolds 23 , generating multi-enzyme pathways with the ability to modulate intermolecular interactions and the local environment [24][25][26][27][28][29][30] .…”

Sequential growth of long DNA strands with user-defined patterns for nanostructures and scaffolds

“…Extended DNA nanostructures have shown promise as structural scaffolds that can organize patterned arrays of cargo, with potential in plasmonics 25 , catalysis 26,27 , artificial light harvesting 28 and nanoelectronics 29,30 . These applications require fine control over the placement and order of specific functional groups, which can be challenging at the nanometre scale.…”