Part:BBa_K3806008

Theophylline-binding aptazyme

Positive control for DRIVER

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Ligand-dependent self-cleaving ribozymes, also known as aptazymes, have emerged in recent years as valuable tools for controlling gene expression [2]. Therefore, aptazymes present a large number of potential applications, ranging from disease diagnosis, prognosis, or treatment to detecting small pollutants in the environment. Aptazymes can be programmed to respond to a wide range of small-molecule ligands with high sensitivity and selectivity. Newly developed methods such as DRIVER (de novo rapid in vitro evolution of RNA biosensors) enable rapid, automated, and multiplexed engineering of aptazymes sequences to diverse small molecules [1].

In the study of Townshed et al. [1], DRIVER was used to evolve a theophylline binding aptazyme that undergoes self-cleavage in the absence of theophylline and remains uncleaved in its presence (Fig. 1). The TU Delft iGEM team 2021: (i) characterized the cleavage of this aptazyme using a Urea-PAGE gel following a co-transcriptional cleavage assay. (ii) showed the value of this part when used as a positive control, and (iii) proved that this part (BBa_K3806018) can be used to regular gene expression in vitro (BBa_K3806014 and BBa_K3806016).

Fig. 1 Theophylline-binding aptazyme (A) 2D structure, and (B) predicted 3D structure. The structure of the aptazyme resembles that of the sTRSV hammerhead ribozyme. It is expected that the binding of the ligand to specific sequences within the large loop (30 nucleotides), affects the interactions with the smaller loop (7 nucleotides), hindering self-cleavage The cleavage site is indicated with a red arrow.

References

1. [1] Zhong, G., Wang, H., Bailey, C. C., Gao, G., & Farzan, M. (2016). Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells. eLife, 5, e18858.
2. [2] Townshend, B., Xiang, J. S., Manzanarez, G., Hayden, E. J. and Smolke, C. (2021). A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors. Nat Commun, 12, 1437.