To prevent and control the spread of antibiotic resistance, thousands of environmental inspectors in China are sent out to measure antibiotic residues in the environment, which is important for antibiotic-abuse surveillance. To make the process more convenient, we aim to develop a portable and rapid-response method to detect antibiotic residues on-site . After paying a great effort into the literature, we found that aptamer is a great tool to detect small molecules including antibiotics1. Aptamer for kanamycin, a kind of antibiotics, has been created and reported to have the strong binding activity 2,3. Aptamers are nucleic acids which are similar to antibodies but cheaper and more flexible. However, it has several other advantages, one of which is that they have the ability of switching their structures upon the recognition of the specific target. Using screening methods like SELEX, specific aptamers with strong binding activity will be sorted out. For example, kanamycin aptamer would form a G-quadruplex upon the binding of kanamycin.

 

Now we have the tool for antibiotic binding. But aptamer itself can not give out signals, how could we use it in antibiotic measuring? To achieve this, we need to combine aptamer with our Cas12a reporting module. We immediately head for the literature for inspiration. Luckily, we found a solution can probably be the bridge to combine aptamer and Cas12a-reporting module4. They found an oligonucleotide which could base pair with the aptamer. It is not until the existence of small molecules that the oligonucleotide can be recognized by Cas12a and give off signal. Inspired by this work, we set about developing a novel system targeting antibiotic residues combining these two powerful tool. Here is how it works.

 

Figure 1.Workflow of the system in the literature, adapted from [4].

 

We designed two aptamers containing the structure-switching sequence to lock another oligonucleotide called activator DNA. Since it is locked, it cannot activate Cas12a’s cleavage. But once aptamers have recognized antibiotics, the activators will be released and can then then serve as the target DNA to activate Cas12a. Then as we introduced in Proof of Concept, the Cas12a will cleave reporter ssDNA and let the whole system emit fluorescence.

 

In fact, several considerations are involved in the designing process of aptamers and activator DNA since they are not exactly the same as the original aptamer. After referring to the literature⁴ and combining the knowledge we have learned, we summarized the following tips. First and foremost, the structure-switching sequences should be included in the aptamers. Secondly, additional sequences should be added at the front and end of the original aptamer to bind the activator DNA more tightly, which can help reduce leakage. Also, based on the knowledge we knew about Cas12a at that time, the activator should be a double-strand DNA with PAM sequence.

 

However, if the activator is double strand structure, it will be impossible for it to pair with aptamers. How to fix this problem? We returned to the article, and found that the author used single-strand DNA as activator! We did not know who was wrong there. But based on our experience of Cas12a, we did not believe that at first. So, we lay that over and turn to other possible solutions. After several weeks, we did not make any progress in this problem. At the time we almost give up this design, we found out that when Cas12a targets ssDNA, it actually does not need a PAM sequence⁵! This information dispelled our doubts and boosted our confidence in the design of the aptamers and the activator.

 

Figure 2.The design norm of locked activator and modifying aptamer.

 

So, we redesigned the element like Figure 2. It obeys the two rules we mentioned above. The structure-switching sequences should be included in the aptamers. And additional sequences should be added at the front and end of the original aptamer to bind the activator DNA more tightly, which can help reduce leakage. And we named it as Aptamer Sandwich. Unsurprisingly, the results turned out to be extremely good. We did orthogonal experiments concerning the concentrations of aptamer-activator DNA complex and kanamycin. We concluded that, as the concentration of kanamycin increased, the fluorescence intensity increased as well (Figure 3a, b).

 

Figure 3. The characterization results of CESAR-1 system:

(a) The fluorescence intensity over different concentrations of kanamycin, i.e., 0, 170, 340. The concentration of aptamer-activator DNA complex is 12.5nM. RFU refers to relative fluorescence unit.

(b) The bar graph of fluorescence intensity at 166min.

 

To summarize, we designed a brand-new element for aptamer to combine with Cas12a detection system. We set out from our needs, created the designed based on literature information, and finally did experiment to successfully tested it. We are proud of our achievement, and it is a great pleasure that we can provide to future iGEM teams with our independently developed components!

Aptamer Sandwich links: BBa_K3454034, BBa_K3454035, BBa_K3454036.

 

 

Reference

¹ Dunn, M. R., Jimenez, R. M., & Chaput, J. C. (2017). Analysis of aptamer discovery and technology. Nature Reviews Chemistry, 1(10), 76.
https://doi.org/10.1038/s41570-017-0076

² Ma, X., Qiao, S., Sun, H., Su, R., Sun, C., & Zhang, M. (2019). Development of structure-switching aptamers for kanamycin detection based on fluorescence resonance energy transfer. Frontiers in Chemistry, 7(FEB), 1–10.
https://doi.org/10.3389/fchem.2019.00029

³ Xing, Y.-P., Liu, C., Zhou, X.-H., & Shi, H.-C. (2015). Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay. Scientific Reports, 5, 8125.
https://doi.org/10.1038/srep08125

⁴ Xiong, Y., Zhang, J., Yang, Z., Mou, Q., Ma, Y., Xiong, Y., & Lu, Y. (2020). Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets. Journal of the American Chemical Society, 142(1), 207–213.
https://doi.org/10.1021/jacs.9b09211

⁵ Kellner, M. J., Koob, J. G., & Gootenberg, J. S. (2019). SHERLOCK : nucleic acid detection with CRISPR nucleases. Nature Protocols, 14(October).
https://doi.org/10.1038/s41596-019-0210-2