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USING DNA ORIGAMI FOR

A NANOSCALE ELECTROCHEMICAL POSITIONER MODEL

Presented by Team Toehold Conga Nanny

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Abstract
Introduction and Theory

Abstract

For this project, our lab created an electrochemical, folding-based DNA origami model that could one day be used to sense viruses and virus-sized objects. We call the origami we are currently experimenting with a “positioner,” which acts as a model for folding-based biosensor distance measurements. We made 9 different positioners, each with a different angle between the base and the arm. Gel electrophoresis, Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM) images show that we have been successful with our synthesis. We equipped positioners with two redox reporters, anthraquinone (AQ) and methylene blue (MB), and thiols for attachment to gold electrodes in order to measure changes in current with each positioner. Square-wave voltammetry (SWV) of positioner-functionalized electrodes should allow us to see changes in normalized current via peak heights for the reporters, but for now we are only able to see signals from the MB.

Introduction and Theory

Our project’s ultimate goal is to develop electrochemically-active biosensors because they can be used in providing real-time monitoring of various substances that circulate in the blood of living animals[1]. With these sensors, they could potentially be used to monitor drug treatments among patients, identify different types of toxins within their bodies or sense the presence of specific viruses within a person’s body. The structure of electrochemical biosensors includes a monolayer-protected gold electrode that has a DNA-strand bearing a redox reporter that produces an electrochemical signal based on how often the redox reporter’s electrons collide with the gold surface (Fig. 1a)[2]. When the DNA strand binds to a particular target molecule, the strand’s conformation changes, so that the redox reporter on the strand becomes closer to the gold surface (Fig. 1a)[2]. This causes a change in the electron transfer rate between the gold surface and the redox reporter as depicted in the graph below (Fig. 1b)[2].

echempic.PNG

Figure 1. a. A generic electrochemical biosensor. b. Graph of signal-on (increased current) and signal-off (decreased current) as a result of target binding

Although DNA strands have been used as biochemical sensors, they have some limitations. Although DNA strands can detect analytes[3,4], they are unable to detect large targets, such as whole-viruses, due to the size of targeted molecules and the length of the strand that is to bind to the target. Additionally, DNA strands can only hold one or two redox reporters, which limits the range of distances that can be obtained when the strand is bound or unbound to a target molecule. Due to these limitations, we turned to using DNA origami because it is capable of binding to larger target molecules and holding more binding sites than a DNA strand would[5,6]. We would also have more control over the position of the redox reporters and binding sites if we used DNA origami for our sensor, rather than strands[5,6]. These advantages led us to developing a triangle made of DNA origami because a triangle would be able to hold multiple redox reporters and binding sites, and its shape is a lot larger than a strand, which allows it to bind to larger target molecules.

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 Although the triangle had more advantages, it produced inconsistent, low current peaks. Since high current peaks are produced when the redox reporter is close to the gold surface, this indicated that the triangle had issues with getting the redox reporters close enough to the gold surface to produce a good signal response. To fix this problem, we decided that we wanted to develop a different biochemical sensor in the form of a “positioner”. A positioner consists of two origami beams linked by four DNA duplexes as depicted in the image below (Fig. 2a)[7]. We chose this shape because we wanted the bottom beam to hold one fixed dye (AQ-red dot) and the top beam to hold a movable dye (MB-blue dot) that would produce distinguishable signals when tested using electrochemistry[8]. Additionally, we wanted something in between that would allow us to move the beams farther apart or closer together, and the shape of the positioner fits this idea because it allows us to control the angle and distance that we want between the two beams, which then controls the amount of current produced when tested using electrochemistry.

origamistructure.PNG

Figure 2. a. Our modified positioner, side view. The Black Bars refer to pairs of helices, antiparallel arrows refer to the adjustable duplexes. Static Reference redox reporters (red dot) are located on the base beam at the same depth as the alkylthiol linkers (the green wavy lines). These linkers attach the positioner to the gold electrode surface (yellow) and functionalized by a monolayer (thin black wavy lines). The longer arm beam that projects past the hinge bears the Measurement redox reporters (blue dot) that moves as the arm moves. b. Perspective diagrams looking at the positioner at an end view.

Through the process of overnight annealing of the proper DNA strands and dyes, which were Methylene Blue (MB) and Anthraquinone (AQ), we then wanted to test whether our positioner was actually formed and if it would give the proper signals when tested on the potentiostat for electrochemistry. In order to test if the positioner was formed, we ran the produced positioners on agarose gels and scanned them using SYBR Gold Stain. If positioner was properly formed, bands would appear on the gel with the proper mobility and would also be above the plasmid’s bands since the positioner is heavier than the plasmid. Additionally, we also used AFM to see if we could visualize detailed 3-Dimensional images of the positioners and determine whether the structures had formed properly. The gels produced did depict the proper bands; however, AFM did not show high resolution, detailed images of the positioners, so we turned to TEM to see if we could get better images of our positioners on a 2-dimensional scale. The TEM images proved to be successful and allowed us to confirm that we did produce positioners with the proper shape and form.

 

To test our positioners responsiveness to electrochemistry, we used electrochemical cells and gold chips, which act as working electrodes, to test our samples (Fig. 3a).

echemcell.PNG

Figure 3. The components of our research grade electrochemical cell.

For our positioners to perform properly, the voltammogram produced under square-wave voltammetry conditions should show signals for both the MB and AQ dyes on the positioner. Additionally, we should be able to see changes in the current caused by the distance created between the dye on the positioner’s arm beam (MB) and the gold surface depending on the angle and distance we set for each positioner (Fig. 4a). Our expectations are to see a sigmoidal relationship between the dye and the gold surface where, when the dye is close to the gold surface, the current should be very high, and inversely, when the dye is far away from the gold surface, there should be little to no current produced (Fig. 4b).

current graph.PNG

Figure 4. a. The positioner in action. b. Simulated plot of basepairs in connecting helixes versus relative content.

However, when the positioners were tested using electrochemistry on the potentiostat, only the MB dye appears on the voltammogram and AQ dye, which is our reference redox reporter, appears to be missing. It is crucial that both dyes show up on the voltammogram because, otherwise, we cannot confirm what relationship the distance between the dye and the gold surface has with respect to the current produced. Due to this issue with the AQ dye, we have taken a step towards trying Ferrocene as an alternative to Anthraquinone by synthesizing an NH2-bearing oligonucleotide strand with Ferrocene NHS-ester-chemistry that we will incorporate into our future positioner strands to see if this will resolve our issue with AQ[9]. Although we are still far from creating the positioner as a signal-on biosensor for whole viruses, our positioner design does solve the issue we had with the triangle’s floppiness and lack of rigidity, and we have produced positioners that fit our desired design, as well as given decent results based off gel scans, TEM, and electrochemistry.

Goals

Goals

Achieved Goals

Achieved Goals

Synthesis​

Imaging​

Electrochemistry​​ (click here to see result)

  • Resolve AQ peak on SWV​

  • Reduce noise on SWV

Near and Future Goals

  • Show the effects of destapling on the yields and concentrations. 

  • Resolve MB peak on SWV​

  • Flatten baseline on the left shoulder​

  • Overcome issues with AQ

    • Develop Ferrocene NHS Ester Oligos

  • Overcome issues with positioners attaching to the gold surface 

    • Develop Dithiol strands

Future Goals
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