Celastrus orbiculatus and Celastrus scandens differentiation using G-quadruplexesBrooke Gillitzer

IntroductionG-quadruplexes are DNA structures that do not bind in the usual Watson and Crick base pairing.

Watson and Crick base pairing is when cytosine binds with guanine and thymine binds with

adenine to form a double helix structure. A G-quadruplex forms when there is Hoogsteen base

pairing and guanines pair with each other. The DNA sequence has to be rich in guanine to form a

G-quadruplex. G tetrads form from four guanine bases and then the tetrads stack on top of one

another to form a G-quadruplex. Alkali metal ions are located in the middle between two of the

tetrads. Hydrogen bonds and the alkali metal ions stabilize the G quadruplex structure. G

quadruplexes can form from one, two, or four strands of DNA.

G quadruplex can act as a DNAzymes when combined with hemin. DNAzymes are DNA

sequences that have catalytic properties and peroxidase-like activity. The DNAzyme is able to

catalyze hydrogen peroxide (H2O2) mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-

6-sulphonic acid) (ABTS) as seen in figure 1. The Y in figure 1 represents the ABTS. The H2O2

oxidizes the hemin and then the hemin oxidizes the ABTS. When ABTS is oxidized, it results in

the solution turning green in color. The goal of this experiment was to develop a DNA sensor

that used G-quadruplex enzyme activity to indicate the presence of a species specific DNA

sequence.

Figure 1: Image of Peroxidase-like activity of DNAzyme.

A molecular device is a tool that uses individual molecules as its moving and working

parts. One specific type of DNA-based molecular device is called a G-quadruplex.

This summer we specifically choose to try to develop a method to be used to differentiate

between two similar species. The two species that we had chosen were the Celastrus orbiculatus

(oriental bittersweet) and the Celastrus scandens (American bittersweet). The oriental and

American bittersweet species are two vining plant species that are very similar in their

morphology. The only time that the two species would be able to be distinguished by sight is

when they are fruiting. The fruit of the oriental bittersweet grows on the leaf axils and along the

length of the stem. The American bittersweet only has fruit on the terminal end. The capsules of

the fruit are also different colors. The oriental has a yellow coloring while the American has an

orange coloring. The two different morphologies can be seen in figure 2. The two species are

able to create hybrids.

Figure 2: Celastrus orbiculatus and Celastrus scandens fruit comparison.

This experiment included designing different DNA sequences to form the molecular

device for each of the two bittersweet species as seen in figure 4. The input sequence for each of

the bittersweets was designed to be a DNA sequence found in only in that corresponding

bittersweet plant species. The output sequence was designed to be able to form a G quadruplex

from two strands and have peroxidase-like activity. A second output sequence was designed for

the American bittersweet to see if the group of guanines that occur naturally in the plant DNA

would have an effect to form G quadruplexes. The sensor sequence is complimentary to the

output sequence and was designed to be able to bind to the output sequence via the usual Watson

and Crick base pairing. This binding would prevent the output strand from binding with another

output strand and forming a G quadruplex. The sensor sequence was designed also to be

complimentary to the input sequence. There is a section of nucleotides called the “toe-hold” that

hang off the end and do not bind to the output sequences. The input sequence is able to bind to

this area and allow for strand displacement to occur. Strand displacement occurring would mean

that the output sequence would be displaced and allow for the sensor and input sequences to bind

together. The output sequences would then be able to bind with one another and allow for G

quadruplexes to for and for there to be peroxidase-like activity.

Figure 3: Sequences of each of the designed DNA segments.

MethodsOutput sequences

For testing for peroxidase-like activity of each of the output strands 250 µL PO4 buffer, 250 µL

KCl, 400 NaCl µL, 50 µL 0.1% triton, 50 µL hemin and 10 µL H2O2 were mixed together in

distilled water. The solution was then placed into cuvettes and 50 µL ABTS was added to each

cuvette. A blank was measured by placing a cuvette with the mixture into a spectrophotometer at

420 nm. The DNA output sequences were then mixed into a cuvette and a spectrophotometer

records the absorbency of each solution every 30 seconds for 15 minutes.

Sensor sequences

The sensor sequence’s ability to anneal to the output DNA and inhibit peroxidase-like

activity was assembled by mixing 250 µL PO4 buffer, 250 µL KCl, 400 µL NaCl, 50 µL 0.1%

triton, 50 µL hemin and 10 µL H2O2 in distilled water and placed into cuvettes. The output and

sensor DNA sequences were heated in a buffer solution to 90° C and then allowed to cool to 25°

C. The 50 µL of ABTS then was mixed into each cuvette and a blank was recorded using a

spectrometer at 420nm. Next, the DNA sequences were placed into cuvettes and placed in the

spectrometer. The spectrometer recorded the absorbency of each cuvette every 30 seconds for 15

minutes.

Input sequences

The input sequence’s ability to bind to the sensor sequence and allow for there to be

peroxidase-like activity was assembled by mixing 250 µL PO4 buffer, 250 µL KCl, 400 µL

NaCl, 50 µL 0.1% triton, 50 µL hemin and 10 µL H2O2 in distilled water and placed into

cuvettes. The output and sensor sequences were heated in a buffer solution to 90° C and then

allowed to cool to 25° C. The input sequences were not heated. Then 50 µL of ABTS then was

mixed into each cuvette and a blank was recorded using a spectrometer at 420nm. The sensor

and output sequences were then placed into the cuvettes. The input sequences were added next

and then the cuvettes were placed into the spectrometer. The spectrometer recorded the

absorbency of each cuvette every 30 seconds for 15 minutes.

Results

Output sequences

The first experiment involved testing the output strands for peroxidase-like activity. Five

different solutions were tested. The first solution contained no DNA as a control for background

oxidation. Another solution contained EAD2 sequence. The other three contained the three

output sequences that were designed as seen in figure 3. Figure 5 shows the absorbance of each

of the solutions containing a different output sequence at 420nm over a period of 15 minutes.

Figure 4: The absorbance of ABTS being oxidized at 420 nm over a period of 15 minutes

Sensor sequences

The second experiment involved testing the sensor sequences to see if they would bind to the

complementary output sequence and inhibit the peroxidase-like activity. Five different solutions

were tested. One solution contained no DNA as a control for background oxidation. Another

solution contained just the oriental output sequence. The next solution contained the oriental

output sequence and the complimentary oriental sensor sequence. The American output sequence

was also tested alone in a solution. The last solution had both the American output sequence and

the complimentary American sensor sequence. Figure 5 shows the absorbance of each of the

solutions containing a different output sequence at 420nm over a period of 15 minutes.

Figure 5: The absorbance of ABTS being oxidized at 420 nm over a period of 15 minutes

Input sequences

The third experiment involved testing the input sequences to see if it would displace the output

sequence and allow peroxidase-like activity. There were five different solutions that were tested.

One contained no DNA as a control for background oxidation. The next contained just the

American output sequence. Another contained both the American output sequence and its

complimentary sensor sequence. The next two solutions contained the American output, sensor,

and input sequences; one solution having 5 µL of input and the other containing 10 µL of the

input sequence. Figure 6 shows the absorbance of each of the solutions containing different

sequences at 420nm over a period of 15 minutes.

Figure 6: The absorbance of ABTS being oxidized at 420 nm over a period of 15 minutes.

DiscussionIn the first experiment when testing the output sequences for peroxidase-like activity, EAD2 was

used as well as the output sequences that were designed. EAD2 is a well-known G-quadruplex

sequence to compare peroxidase-like activity to. From the results in Figure 5, we can see that

EAD2 has the greatest absorbancy levels. From this we can say that EAD2 solution has the most

G-quadruplexes forming. The higher the absorbancy levels means that the solution has turned a

darker green compared to the others. Figure 5 also shows that the Oriental output sequence had

similar absorbancy levels compared to the American output1 sequence. These sequences had

higher absorbancy when compared to the No DNA solution so each are showing that those

sequences form G-quadruplexes. The American output2 sequence showed similar absorbancy

levels to the solution with no DNA. We decided to no longer use the American output2 sequence

going forward in the experiments due to having lower peroxidase-like activity.

The next experiment was to test if there was inhibition of G-quadruplexes forming when

the output sequences were mixed with the corresponding sensor sequences. Each of the two

output sequences were put into a solutions without any other DNA sequences. This was done to

compare the G-quadruplex formation to the output sequence mixed with the corresponding

sensor sequence’s inhibition of G-quadruplex formation. Figure 6 shows that when the output

sequences were added with the sensor sequences, there was a reduced amount of absorbance

compared to the output sequence alone. This means that there was some inhibition of the output

sequences when combined with the sensor sequences.

The final experiment of testing if the input sequenced allowed the output sequences to be

displaced from the sensor sequences and allow for the output sequences to form G-quadruplexes

is still being worked out. These tests had a variety of results. There was peroxidase-like activity

from the input sequences added to the output and sensor sequence solutions shown in figure 7.

Figure 7 data was the only trial that showed these results. The other trials showed peroxidase-like

activity from the input sequences added to the output and sensor sequence solutions.

Future research needed to be done for this project include continuing the input sequences

for the displacement of the output sequences and allowing for peroxidase-like activity. Other

future directions for this project could include testing different concentrations of the reagents

used to optimize the reaction. Another direction could be to look at how long strand

displacement occurs. We also would like to how one change in a nitrogenous base would affect

the experiment.

AcknowledgementsI would like to thank Peter Kuhn, PhD, Assistant Professor at Edgewood College who is the

principal investigator for his time and guidance on this project. I would also like to thank Shad

Wenzlaff, who is the director of the summer undergrad research.

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