Difference between revisions of "Part:BBa K3938001"

Latest revision as of 23:37, 19 October 2021

ssDNA，an aptamer for 3O-C12-HSL.

This aptamer is a single-stranded DNA that can specifically bind to 3O-C12-HSL. It was tested by SYBR GreenⅠ（SG）fluorescence method, which indicated it can specifically bind to 3O-C12-HSL.
The aptamer was originally from a paper that found 3 aptamers (78 bp each) for 3O-C12-HSL and C4-HSL respectively(Zhao et al., 2013). We used one of 3O-C12-HSL aptamers as our aptamer, the first step was to test the binding ability of the aptamer, so the SYBR GreenⅠ（SG）fluorescence method was used.
The principle of the method was that SG acts as a DNA fluorescent dye that did not carry a fluorescent signal of its own (Abraham et al., 2018; McKeague et al., 2014). When bound to the aptamer, it fluoresced because it was embedded in the secondary structure of the DNA. Thus when aptamer iwas bound to the SG dye, green fluorescence was produced when stimulated by excitation light（with the excitation Wavelength 485 nm and emission wavelength 535 nm). However, when the target small molecule was 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.
Unfortunately, we did not observe a significant fluorescence qunenching using the aptamer, so we redesigned the aptamers and cut off the constant region of the aptamer, leaving only 35 bp of the aptamer left, which was the sequence that actually binded to the 3O-C12-HSL. Then we also used the SG fluorescence method to test the new aptamer .
We set the concentration of the aptamer as 0, 100, 200, 400, 600, 800 and 1000 nM. As can be seen from figure 1, compared to the fluorescence intensity when the 3O-C12-HSL was 0， the adding of 3O-C12-HSL showed good fluorescence qunenching effects(figure 1). Demonstrating the usefulness of the aptamer binding 3O-C12-HSL.

The fluorescence quenching effect by 3O-C12-HSL using SG fluorescence method

Reference:
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.
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.
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

Sequence and Features