科研之友
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摘要
Hammerhead aptazymes have been widely used to regulate gene expression, and to maintain the tertiary structure interaction between Loop I and Loop II, which is crucial for highly efficient self-cleavage. The existing hammerhead aptazymes are based on stem III or stem I; hammerhead aptazymes based on stem II have not yet been reported. To increase the diversity and universality of hammerhead aptazymes, we explored hammerhead aptazymes based on stem II. By rationally designing the communication module of hammerhead aptazymes based on stem II, and based on the toxic protein ibsC reporting system combined with the growth of the host, we speculate on how hammerhead aptazymes respond to the regulation by small molecules. In this study, 10 types of communication modules with different lengths and base complementary pairing degrees were designed. Among them, the hammerhead aptazymes corresponding to seven kinds of communication modules could not self-cleave, and two types could self-cleave, but they were both not regulated by theophylline. Finally, we have obtained a hammerhead aptazyme T4 based on stem II, whose communication module consists of four pairs of entirely complementary base pairs, which might make it easier to obtain small molecule-dependent hammerhead aptazymes. In the presence of theophylline, the host including T4 grew, while in the absence of theophylline, the host could not grow, which indicated that the self-cleaving activity of hammerhead aptazyme T4 was regulated by theophylline, and successfully achieved using small molecules to control gene expression in Escherichia coli. The T4 is a theophylline-dependent hammerhead aptazyme based on stem II, which indicates that hammerhead aptazymes could be developed based on stem II. Besides, with the trans-cleaving potential, T4 is expected to further be developed into a novel tool that uses small molecules to regulate the expression of endogenous genes.
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