Team:GCGS China/Engineering
Engineering success
Introduction
To verify the functionality of two 78 bp aptamers targeting C4-HSL and 3O-C12-HSL as mentioned in the literature(Zhao et al., 2013), we used the SG fluorescence method as well as the FAM fluorescence quenching method.
1. SYBR GreenⅠ （SG）fluorescence method
Design
This method has been used several times as a label-free test and is based on the principle that SG acts as a DNA fluorescent dye that does not carry a fluorescent signal of its own (Abraham et al., 2018). When bound to the aptamer, it fluoresces because it is embedded in the secondary structure of the DNA. Thus when aptamer is bound to the SG dye, green fluorescence is produced when stimulated by excitation light（with the excitation Wavelength 485 nm and emission wavelength 535 nm). However, when the target small molecule is added to the system, the specific binding of the small molecule to the aptamer results in competition with the SG for the binding site, and as a result, the fluorescence value of the system decreases(figure 1).
Fig 1.SYBR Green I fluorescence-based aptasensing scheme
(McKeague et al.,2014)
The sequences of the original aptamers were as follows:
- 3O-C12-HSL-apt1: 5'-GCAATGGTACGGTACTTCCCGGGGCCCGCTTCTGGTGCGGTGTACTAGTGACCGCAAAAGTGCACGCTACTTTGCTAA-3’
- C4-HSL-apt1: 5'-GCAATGGTACGGTACTTCCCGGGGCCCGCTTCTGGTGCGGTGTACTAGTGACCGCAAAAGTGCACGCTACTTTGCTAA-3’
Analysis and learn
The ability of the 78 bp (base pairs) long aptamer to carry out fluorescence quenching was not effective（figure 2）; we began to discover why. It was later considered that even though the small molecule was bound to the long aptamer sequence, the constant region at both ends would not be affected by the small molecule and would still bind to the SG dye and produce fluorescence, which may be the reason for our un-ideal experiment result. Therefore, we removed the constant regions from two ends of the 78bp long aptamer. After further design and refinement, the new re-designed aptamer is 35bp long ( McKeague et al., 2014; Sarpong & Datta, 2012; Yi et al., 2019).
Fig 2.The fluorescence quenching effect by C4-HSL(left) and 3O-C12-HSL(right）using 78bp aptamers
Redesign and build
To prove that our redesigned sequences are correct and usable, we synthesized new aptamers with only intermediate functional sequences according to the modified sequences, which are as follows（BBa_K3938001 BBa_K3938002）：
The 78bp and 35bp aptamers reacted with SG fluorescence separately and the 78bp aptamer was found to have a higher fluorescence value than the 35bp sequence. This verified that the constant region itself also caused the SG to fluoresce(figure 3).
Fig 3.SG-bound fluorescence values for different lengths of aptamer
(where C4 represents aptamer of C4-HSL and C12 represents aptamer of 3O-C12-HSL)
Test
Next, we tested the fluorescence intensity of small molecules (C4-HSL, C12-HSL) at concentrations of 0nM, 200nM, 400nM, 600nM, 800nM, 1000nM.The experimental results further demonstrated the superiority of the modified aptamers. Regardless of the concentration of small molecules, the SG fluorescent dye was successfully competed off by the small molecules bound to the aptamer, resulting in a significant decrease in fluorescence intensity(figure 4).
Fig 4. The fluorescence quenching effect by C4-HSL(left) and 3O-C12-HSL(right）using 35bp aptamers
2. FAM labeled aptamer-based fluorescence structure switching assay
Design
Besides the SG fluorescence method, we also used another structure switching fluorescence to verify the aptamers can specifically bind to C4-HSL and 3O-C12-HSL. Aptamers with FAM-labeled at 5' end were designed and anti-aptamers which were complementary with the sequence close to the 5′ end of the aptamer were also designed with black hole quencher(BHQ1) labeled at 3' end. In the absence of AHL molecules, the anti-aptamers would be hybridized with the aptamers, causing fluorescence quenching. In the presence of AHL molecules, the small molecules would bind to aptamers, competing with anti-aptamers, thus leading to a fluorescence recovery(Li et al., 2018).
Fig 5. The mechanism of aptamer based structure switching fluorescence assay
The 78bp aptamer was first tested for anti-aptamer length in this project to confirm the appropriate sequence length. The length of the complementary sequence was set to 100%, 50%, 33% of the original length of the aptamer respectively.
(Note: The aptamer for this procedure was derived from the same literature as the aptamer in the SG experiment, which screened three aptamers for C4-HSL and 3O-C12-HSL respectively. Apt1 was validated to have the smallest kd value among the documents, so all apt1 was used in the later experiments, and because of the similarity of these aptamers (because of the high similarity of these aptamers, the same idea can be used in later designs).
- 1.3O-C12-HSL-apt2：5'-FAM-GCAATGGTACGGTACTTCCCGCCGCCGCGTTCTCATCGCGTGATGTAGTGTCCGCAAAAGTGCACGCTACTTTGCTAA-3'
- 2.C12-anti1： 5'-TTAGCAAAGTAGCGTGCACTTTTGCGGACACTACATCACGCGATGAGAACGCGGCGGCGGGAAGTACCGTACCATTGC-BHQ-3'
- 3.C12-anti2： 5'-TTAGCAAAGTAGCGTGCACTTTTGCGGACACTACATCAC-BHQ-3'
- 4.C12-anti3： 5'-TTAGCAAAGTAGCGTGCACTTTTGCG-BHQ-3'
- 4.C4-HSL-apt2: 5'-FAM-GCAATGGTACGGTACTTCCCGGGGCGGGCTGTCATGCCCATCCTACCGTGACCGCAAAAGTGCACGCTACTTTGCTAA-3'
- 5.C4-anti1: 5'TTAGCAAAGTAGCGTGCACTTTTGCGGTCACGGTAGGATGGGCATGACAGCCCGCCCCGGGAAGTACCGTACCATTGC-BHQ-3'
- 6.C4-anti2: 5'-TTAGCAAAGTAGCGTGCACTTTTGCGGTCACGGTAGGAT-BHQ-3'
- 7.C4-anti3: 5'-TTAGCAAAGTAGCGTGCACTTTTGCG-BHQ-3'
Learn and redesign
After experimental measurements, it was found that when the complementary sequence was half of the aptamer sequence, the whole system showed a more obvious fluorescence quenching effect and re-brightening. Based on the results above and the SG fluorescence method, we decided to use the aptamers without constant regions and anti-aptamers having half the length of the aptamers. The design would make the aptamer synthesis more simple and accurate, in addition leading to the better feature of aptamers. Thus we anticipated the results would show a significant fluorescence quenching caused by anti-aptamers and fluorescence recovery caused by AHL binding.
Build and test
We redesigned the aptamers and anti-aptamers, the sequence were synthed and as follows:
- 3O-C12-HSL apt4: 5'-FAM-CGGGGCCCGCTTCTGGTGCGGTGTACTAGTGACCG-3'
- C12-anti4 5'-CACCAGAAGCGGGCCCCG-BHQ-3'
- C4-HSL apt4: 5'-FAM-CGCTGCCCCCTGTCCTGGGTTAGCTAGCGAGAGCG-3'
- C4-anti4: 5'-CCAGGACAGGGGGCAGCG-BHQ-3'
The apt-4 for C4-HSL and 3O-C12-HSL were FAM modified apt2 for C4-HSL and 3O-C12-HSL respectively.
Using this sequence for FAM experiments, the results of the experiments appear to be better quenched and re-lit, as shown below (C4-HSL results on the left, 3O-C12-HSL results on the right（figure 6).
Fig 6. The fluorescence qunenching and recovery of FAM modified aptamers caused by anti-aptamers and AHL
References:
- Abraham, K.M., et al., In Vitro Selection and Characterization of a Single-Stranded DNA Aptamer Against the Herbicide Atrazine. ACS Omega, 2018. 3(10): p. 13576-13583.
- Li, Y., L. Sun, and Q. Zhao, Development of aptamer fluorescent switch assay for aflatoxin B1 by using fluorescein-labeled aptamer and black hole quencher 1-labeled complementary DNA. Anal Bioanal Chem, 2018. 410(24): p. 6269-6277
- McKeague, M., et al., Selection and characterization of a novel DNA aptamer for label-free fluorescence biosensing of ochratoxin A. Toxins (Basel), 2014. 6(8): p. 2435-52.
- Sarpong, K. and B. Datta, Nucleic-Acid-binding chromophores as efficient indicators of aptamer-target interactions. J Nucleic Acids, 2012. 2012: p. 247280.
- Yi, H., et al., Fluorometric determination for ofloxacin by using an aptamer and SYBR Green I. Mikrochim Acta, 2019. 186(10): p. 668.
- Zhao, Z.G., et al., Screening and anti-virulent study of N-acyl homoserine lactones DNA aptamers against Pseudomonas aeruginosa quorum sensing. Biotechnology and Bioprocess Engineering, 2013. 18(2): p. 406-412.