Team:OUC-China
Firstly, a library that has 1w sequences is built. Next, analyze the secondary structure of these sequences with the rnafold function in MATLAB, iterate over ten rounds, each round filter out the sequences with free energy above average, and finally get a new library.
At this time, the remaining aptamers can already form a stable secondary structure and these aptamers are utilized for virtual screening with GABA by autodock vina Given that there are many other small molecules in the cell, in order to ensure the specificity of the aptamers, it is necessary to conduct reverse sieve by some other small molecules in the cell. Fig 2 A. The structure of ATP. B-D the structure of the small molecules utilized in reverse sieve. According to the results of positive sieve and reverse sieve, select the aptamer with lower positive-sieve binding energy and higher reverse-sieve binding energy as an alternative aptamer. From fig3, it can be seen that the aptamer can indeed interact with the ligand GABA. What’s more, from the result of molecular dynamic(fig4), it is clearly that the aptamer can bind GABA as the RMSD value tends to converge. Then the aptamer will be utilized to retrofit into a riboswitch and RNA transporter. Through the literature[2], a riboswitch that can function in eukaryotes has been discovered on the principle of strand-displacement information transmission mechanism, which is used to build escape switch in this project. Add some sliding sequences after the ribozyme and aptamer sequences after the sliding sequences. The secondary structure of RNA is analyzed by RNAforder(fig5). Utlizing another suicide switch involved in the literature[2], the principle of which is the helix-slipping information transmission mechanism, the filtered aptamer sequence is added to the loop of the switch to construct the required switch. This switch ensures that the suicide protein YopE is expressed when the ligand concentration in engineered bacteria decreases. To ensure its feasibility, the secondary structure of suicide switch RNA is analyzed by RNAforder(fig6). In view of that, it adds the whole sequence of the aptamer, there is no need to worry about its binding domain changing. According to the literature[3], the modification of aptamers into RNA transporter cannot directly add all the sequences of GABA aptamers into the backbone of the RNA transporter aptamer. It can only insert the binding region into the center of the backbone of RNA transport aptamer. To ensure its feasibility, the secondary structure of the aptamers is firstly analyzed(fig7) According to the comparison between the secondary structure and the that in the literature, it is found that the yellow area was consistent with the one in literature which means the structure is correct. According to fig8, it is proved this structure is definitely correct, and it is built and test in wet experiments. However, after wet experiment verification, it is found that although the aptamer can indeed bind to ligand GABA, its binding ability is very weak Therefore, we carry this idea to design another aptamer that has higher binding ability.
The docking result is in fig(9). Follow the above process again to build the appropriate components.
After testing in wet experiment, it is found that the performance of the redesigned aptamer is better than the previous one(fig10).According to fig10, it is presented that, at the same concentration, the fluorescence value of GABA2 is significantly higher, and it can be seen from this that the binding ability of GABA2 is stronger than that of GABA1. And after 7.5 µm , the fluorescence value of GABA2 tends to converge, while the fluorescence value of GABA1 continues to increase, which can indicate that the binding ability of GABA1 is weaker than GABA1.
Aptamer Model
Build the library
Positive sieve
reverse sieve
The GABA aptamer sequence library can be obtained through the above process. In this project, RNA transporter and riboswitch based on aptamer are utilized to function in the circuit. Therefore, autodock 4.0 is used to perform a specific analysis of each alternative aptamer.
Given that the binding pocket is unknown, the entire aptamer is set as binding pocket, then the docking analysis is performed by Genetic Algorithm.
The riboswitch before the weak promoter
The riboswitch before YopE
RNA transporter
Although there is no problem with the secondary structure, it does not rule out that its binding domain has changed after such modifications. Whether the structure can bind to the ligand still needs to be verified(fig8).
