Via Research Recognition Day Program VCOM-Carolinas 2025

Biomedical Research

Construction of a DNA origami nanoreactor to study enzyme cascades Tanner Brinager, Grey Coleman, and Patrick D. Ellis Fisher, Ph.D. Department of Biology, Wingate University

Abstract

Results

Conclusions

A

B

Design : A 26-helix bundle DNA origami structure was designed using caDNAno software (Figure 1). The multicolored staple strands represent positions where enzymes can be attached to the nanoreactor in future studies. The atomic model shows the dimensions and scaled size of the projected nanoreactor (Figure 2A ). Cando’s molecular dynamics stability simulation showed high stability within the center (blue) and slight instability (red) towards the edges (Figure 2B). The instability towards the edges is due to the limited number of staple strand crossovers. Assembly : The self-assembly process depends on specific conditions such as the folding reaction time, temperature, and MgCl 2 concentration (Figure 3). Thermal annealing gradients and buffer conditions are especially important to the folding of these structures. Magnesium cations enable folding of the origami by neutralizing the electrostatic repulsion between sugar-phosphate backbones. Starting with a standard 36-hour annealing protocol, various magnesium chloride concentrations were tested. Figure 3A shows that folding buffer containing 12 mM MgCl 2 produced a single, crisp, fast-running band on the agarose gel, indicating a compact structure, while avoiding the aggregation present at high concentrations, when the DNA structures do not migrate out of the wells. To abbreviate the folding time, a brief, 2-hour isothermal folding was protocol was tested. It was determined that a 2-hour fold at 59°C, 61°C, or 62°C, using 12mM MgCl 2 gave the best results, according to gel electrophoresis analysis (Figure 3). Structural Validation : The folded nanostructure produced from a 2-hour fold at 62°C was excised from the agarose gel and purification (Figure 4). Observation was attempted with Transmission Electron Microscopy (TEM) but was unsuccessful. The suspected error is due to grid preparation where the nanoreactor is not properly sticking to the carbon-coated copper surface, preventing observation. Future Directions The immediate next step of this project is to validate the structures by transmission electron microscopy (TEM, Figure 4) or atomic force microscopy (AFM). Atomic Force Microscopy (AFM) is an observation technique taking a more mechanical approach, using a cantilever and laser to topographically map the nanostructure surface. This technique eliminates the use of a grid, which should eliminate error and allow successful observation. The nanoreactor contains twenty different locations at which an enzyme can be placed on the structure (color-coded staple strands in Figures 1B and 2A, attachment sites shown as black circles below). Enzymes will be attached to complementary ssDNA “anti - handles” and hybridized onto these handle attachment points like a peg board. Selecting a specific pair of attachment sites with distinct DNA sequences, two consecutive enzymes in a biochemical pathway can be precisely arranged, and varying spatial arrangements of enzymes can be tested. Closer placement of enzymes would theoretically increase reaction kinetics. Smaller distances between enzymes could speed up diffusion limited reactions by shuttling intermediates. One candidate reaction sequence would be the synthesis of ethanol from pyruvate, involving the enzymes pyruvate decarboxylase and aldehyde dehydrogenase. When placed in close proximity, the reaction kinetics should increase, allowing for a faster and more efficient production of ethanol. Ultimately, these enzyme-functionalized DNA nanoreactors will help study and accelerate biochemical reactions.

DNA origami is a recent technique used to produce complex nanostructures out of DNA. DNA is a double-stranded molecule that folds due to hydrogen bonds between nitrogenous bases on the two strands. The base pairing rules that regulate this folding are well understood, so by designing short single- stranded DNA (ssDNA) “staple” strands that bind to designated regions on a longer “scaffold” strand, a DNA origami structure can be formed. In this study, a “ nanoreactor ” structure was designed using the computer program caDNAno and visualized using the program Cando. 88 staple strands were combined with a 3024 nucleotide long ssDNA scaffold and allowed to fold under thermal annealing. Optimal conditions for folding were tested by varying the thermal gradient and concentrations of MgCl 2 . Proper folding of the DNA was evaluated using agarose gel electrophoresis, verifying that a single, compact DNA nanostructure was generated. The ultimate purpose of this study is to test the impact of enzyme proximity on reaction rates in enzyme cascades. Enzyme cascades are found in many biological systems, but the effects of enzyme spacing upon reaction rates are not well understood. This will be tested by attaching enzymes to different locations on the nanoreactor; reaction kinetics of the various arrangements of enzymes could be compared to determine optimal spacing for enzyme cascades. With this information, DNA origami nanoreactors could serve as a base to accelerate biochemical reactions that depend upon enzyme cascades.

Introduction

Deoxyribose nucleic acid (DNA) is the fundamental molecule that stores genetic information in biological systems. The molecule is double-stranded and folds according to strict base pairing rules; hydrogen bonding between nitrogenous bases (adenine to thymine, guanine to cytosine) holds the double-helical structure together. In recent years, nanotechnology has been able to use the nature of DNA to manufacture nanostructures through a technique known as DNA origami. In DNA origami, the function of DNA is not to store genetic information, but to assemble into nanostructures that may be used for other purposes. A long ”scaffold” strand and many short “staple” strands are mixed together in a “one - pot” reaction in which the designed structure will self -assemble (Rothemund). Short, single- stranded oligonucleotides, called “staples,” bind to complementary regions on a long, circular scaffold strand, pinching it to produce a three-dimensional folded structure. The complementary base pairing between the scaffold and staple strands is favored by the free energy of hybridization, with the DNAdouble helices folding into their lowest energy state to generate the intended structure. This method can be used to cheaply create nanostructures using a bottom-up assembly approach that is much simpler to perform than other nanofabrication techniques. This study aimed to create a “nanoreactor” with various positions on which enzymes could be attached. In the present study, a DNA origami “nanoreactor” was designed, constructed, and analyzed, and will be functionalized with enzymes to study the distance-dependent kinetics of enzymatic cascades. By placing the enzymes that make up an enzyme cascade into differing sites on the nanoreactor structure, varying enzyme proximity could be compared to experimentally determine the importance of enzyme proximity on reaction kinetics.

Figure 1. caDNAno design. A : Cross-section of DNA origami nanoreactor design. Each yellow circle represents a DNA double helix, arranged on a honeycomb lattice. B : Full nucleotide map of the caDNAno design. Each circle represents a DNA double helix, arranged from the first nucleotide on the left to the final nucleotides on the right. A 3024 nt circular scaffold strand, shown in blue, is routed through all helices. Short staple strands, shown in grey and multiple colors, are designed to cross over between helices to provide structural support for the 3-dimensional folded structure.

35 nm

A

23 nm

6 nm

53 nm

B

Figure 2. Cando structural predictions. A : Atomic model of DNA nanoreactor, including designed dimensions. Blue represents scaffold strand; grey and multicolor represent color-coded staple strands, where proteins could be attached. B : Molecular dynamics stability simulation. Color scale represents root mean square fluctuation (RMSF) values, showing low flexibility regions (bluest, ~0.25 nm) to high flexibility regions (reddest, ~1 nm).

Dey et al .

A: Magnesium Titration

B: 2 hour ‘Magic Fold’

Methodology

Potential enzyme attachment sites on the nanoreactor, corresponding to the 3’ ends of select staple strands.

Fu et al .

Design and Structural Prediction : The structure for the nanoreactor was designed using caDNAno software (Douglas et al .). Structural dimensions were chosen and laid onto a honeycomb lattice cross-section of DNA double helices (Figure 1A). The scaffold strand was routed through all helices in the cross section to form a single shape. Staple strands were then routed and broken into short fragments around 30 – 42 nucleotides in length. Molecular dynamics simulations of the design were performed using the online software Cando (Kim et al .). This program performs analysis on DNA twist, bend, and stretch stiffness to model the structural stability of an origami nanostructure (Figure 2). Assembly : A 3024 nt scaffold strand (p3024), a gift from collaborators at Yale University, was combined with a 6-fold excess of 88 distinct staple strands, synthesized by IDT, in a “one - pot reaction” and allowed to base pair by thermal annealing to assemble t he designed DNA origami nanoreactor. The thermal ramp and buffer conditions were tested to optimize the efficiency of the folding reaction. Folding efficiency was tested in 1x TE buffer containing MgCl 2 concentrations ranging from 6 mM to 20 mM. A standard 36 hour slow annealing protocol, from 80 – 15 ° C, was used to test the optimal cation concentration for nanostructure folding. During the thermal annealing process, the DNA was heated and slowly cooled, allowing each strand to find its complement. Using the 2-hour “Magic Folding” protocol, structures were folded at a constant temperature (46, 47, 49, 52, 56, 59, 61, or 62° C) and analyzed to determine which condition produced the best-folded nanoreactor. Gel Analysis and Purification : Proper folding of the DNA under each condition was evaluated using agarose gel electrophoresis to verify that a single, compact DNA nanostructure was generated. Folding reactions were run on a 1.5% agarose gel in 0.5x TBE + 10mM MgCl 2 + 0.5 µg/mL EtBr and run at 60V for 1.5 – 2hr and visualized with UV light (Figure 3). To purify folded structures, bands of well-folded nanoreactor products were excised from the gel (Figure 4 ), diced, and purified using Freeze ‘N Squeeze columns (BioRad). Gel pieces were frozen at -20 ° C for 5 minutes and spun at 13000 x g through the column to separate agarose fibers from a solution containing the purified nanoreactors. TEM Sample Preparation : Samples for transmission electron microscopy were prepared on Formvar/carbon coated copper grids (Ted Pella, Inc.) using uranyl acetate (1% in 1x TE + 10 mM MgCl 2 ) as a negative stain (Figure 4).

References

Dey S, Fan C, Gothelf KV et al . DNA origami. Nat Rev Methods Primers. 2021 ; 1(13). Douglas SM, Marblestone AH, Teerapittayanon S, et al . Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res . 2009;37(15):5001-6. Fu J, Oh SW, Monckton K, et al . Biomimetic Compartments Scaffolded by Nucleic Acid Nanostructures. Small . 2019. Kim DN, Kilchherr F, Dietz H, Bathe M. Quantitative prediction of 3D solution shape and flexibility of nucleic acid nanostructures. Nucleic Acids Res. 2012;40(7):2862-2868. Rothemund PWK. Folding DNA to create nanoscale shapes and patterns. Nature . 2006;440:297-302. Acknowledgements

Figure 3. Optimization of DNA origami folding reactions. 1.5% agarose gels in 0.5x TBE + 10mM MgCl 2 + 0.5 µg/mL EtBr were run at 60V for 1.5 – 2hr and visualized with UV. A : Magnesium titration. To form tightly-compact DNA structures, divalent cations are used to neutralize charge repulsion. A range of MgCl 2 from 6 – 20 mM was tested in 36-hour folding reactions, which were then subjected to gel electrophoresis. B : 2-hour “magic fold” reactions were performed at different temperatures in the presence of 12 mM MgCl 2 . In both gels, a DNA ladder and p3024 scaffold strand were run to compare band migration. Bands below 0.5 kb show excess staples added during the folding reactions.

2-hr Fold at 62 ° C

p3024 scaffold

Ladder

10 kb 3

1 0.5 1.5 2 1.2

excised band

purified nanoreactor

negative-stain TEM grid

The authors would like to thank Dr. Chris Dahm and Cyndi Dahm for funding this project through the Wingate University LED Summer Research Grant. Previous undergraduate students in the lab, Paris Brewster, Aliyah Goldsmith, and Alyssa Jarvis, contributed to study design and data collection. The p3024 scaffold DNA was graciously provided by Drs. Chenxiang Lin, Qingzhou Feng, and the Lin lab at Yale University School of Medicine, Nanobiology Institute.

Figure 4. Gel purification of folded nanostructure. Nanoreactor monomer band was excised and purified away from agarose fibers. Purified nanoreactors were applied to Formvar/carbon coated copper grids for negative-stain transmission electron microscopy (TEM).

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