Properties

Introduction

The basic part we are presenting is a component of a cell-free biosensor which is designed to determine whether a liquid sample contains above a certain concentration of deltamethrin or not.

The aptamer plays a key role in the detection process since it is the one that performs the recognition and binding of deltamethrin and then triggers a hybridization cascade with the final objective being the qualitative detection of the pesticide through a chromatic change in the solution.

Short description

Because of its selective sequence it has a high affinity for the target molecule and thus is able to identify deltamethrin and bind with it. After this binding, its tertiary structure of the selective sequence alters causing the S segment to dissociate from its complementary chain (cdAPT). As a free molecule, it can binds to L1 chain due to the complementarity of its S region with the a*b*c* region of the L1 chain, thus forming the S-L1 double-stranded DNA molecule essential for the continuation of the detection process as shown below.

Picture 1: Detection system mechanism following deltamethrin-dAPT binding

EXPERIMENTAL CYCLE 1 (DOCKING)

First, we tested its selectivity using the MOE docking software, which accounts for its tertiary structure change. In more detail, we used dAPT’s 3D structure in .pdb file format together with the 3D structures of deltamethrin and l-cyhalothrin, also in .pdb file format. We entered the aptamer structure separately with deltamethrin and l-cyhalothrin to compare their binding scores and observe whether its selectivity for l-cyhalothrin compares with that of deltamethrin. Essentially, l-cyhalothrin served as a positive control as the selective aptamer sequence was taken from a publication (Yang et al., 2021) that provided a list of highly selective aptamer sequences for detection of l-cyhalothrin (more about the sequence source on the registry page). The binding scores were quite satisfactory as this aptamer sequence had the highest binding score on deltamethrin binding among the thirty sequences we tested, and also had a higher binding score on deltamethrin binding compared to l-cyhalothrin, which means that this aptamer has both the highest affinity of the remaining 30 structures but also has a higher affinity for delta than the target molecule for which it was originally designed.

Picture 2: dAPT 2D structure used to predict the 3D structure of dAPT as described at engineering page.

Picture 3: dAPT 3D structure used in the docking software to determine its binding affinity to deltamethrin.

Picture 4: 3D structure of l-cyhalothrin used in docking.(visualized at PyMol) (left)

Picture 5: 3D structure of deltamethrin used in docking.(visualized at PyMol)

Figure 1: Docking results showing binding scores of the five most stable dAPT-l-cyhalothrin complex (first 5 rows) and dAPT-deltamethrin complex (last 5 rows)

EXPERIMENTAL CYCLE 2 (QCM)

Following the above experimental procedure we tested the aptamer using QCM biosensor , in order to:

1. Confirm docking results and
2. Determine whether the duplex dAPT-cdAPT will dissociate in the presence of deltamethrin (due to deltamethrin binding altering its tertiary structure as mentioned above) thus testing its selectivity to the pesticide

After completing the experiments on the acoustic biosensor and analyzing the results obtained, we were led to the conclusion that despite the small difference in the signals between the aptamers, the aptamer dAPT-11 is the most suitable for our system as well as and to be submitted to the registry since we observed expected, satisfactory results and quite a difference with the control.

Figure 2: Comparison of the signals between the control sensor and the sensor with dAPT11

CONCLUSION

This part, due to the simplicity of application could provide a useful means of detection (standalone or as a part of a multi detection process) for future iGEM endeavours while also enhancing the ever-growing registry section that revolves around aptamer detection, a method that steadily picks up momentum in the scientific community.