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 [1]. Therefore, aptazymes can be used in large number scenarios, 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 a rapid, automated, and multiplexed engineering of aptazymes sequences to diverse small molecules [2].

In the study of Townshed et al. [2], 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) validated the cleavage activity of this aptazyme using a Urea-PAGE gel following a co-transcriptional cleavage assay, (ii) showed the value of BBa_K3806008 when used as a positive control in the setup and course of DRIVER experiments, and (iii) proved that BBa_K3806008 can be used to regulate gene expression in vitro (BBa_K3806010, BBa_K3806014, BBa_K3806015 and BBa_K3806016).

Fig. 1 Theophylline-binding aptazyme (BBa_K3806008). (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.

Experimental results

Cleavage characterization of the thophylline-binding aptazyme

To assess the cleavage activity of the theophylline-binding aptazyme, BBa_K3806008 was fused to the T7 promoter (BBa_K3806011). Transcription reactions were run for 30 minutes at 37 °C, with and without the addition of theophylline. Subsequently, a Urea-PAGE gel was run to visualize the transcription products. The bands corresponding to the uncleaved and cleaved products of the theophylline aptazyme are expected to be located at 109 bp and 89 bp, respectively. It can be observed that the cleavage fraction decreases when increasing theophylline concentration (Fig. 2)

Fig. 2 Urea-PAGE gel for characterization of theophylline aptazyme cleavage (BBa_K3806008) fractions following a co-transcriptional cleavage assay.. Lanes: (Ladder) denatured dsRNA ladder, (1) Negative control: no DNA template. (2) gB-Theo DNA template (without T7 RNA polymerase added to the transcription reaction), transcription of gB-Theo with (3) 0 mM theophylline, (4) 0.5 mM theophylline, (5) 10 mM theophylline.

ImageJ was used to quantify the intensities (I) of the gel bands, and the cleavage fraction (f) was determined by:

A 71 % of cleavage was observed at 0 mM theophylline (Fig. 2, lane 3), 39 % at 0.5 mM theophylline (lane 4), and 4 % at 10 mM theophylline (lane 5). These results confirm the ligand-dependent cleavage activity of BBa_K3806008
.

BBa_K3806008 as a positive control for in vitro engineering of aptazymes

As BBa_K3806008 shows a ligand-dependent cleavage activity, it can be used as a positive control to set up and track the progress during DRIVER [1] experiments. Here, an example of the use of this part as a positive control to set up the DRIVER reaction conditions is presented. To engineer ligand-specific aptazymes using DRIVER, target ligands are supplemented to the transcription reaction, such that functional aptamers co-transcriptionally self-cleave in the absence of ligands, and remain uncleaved in their presence. The buffer in which the ligands are dissolved can potentially interfere with transcription. In this example the interference of the pH of folate solutions at different concentrations was tested (Fig. 5), using BBa_K3806008
as the DNA template for transcription. It was observed that the addition of folate solution adjusted to a pH range of 8 to 9 does not affect transcription, and that cleavage in this range was as well posible. By contrast, adding 1 μl of a more concentrated folate solution dissolved in 1 M NaOH was incompatible with transcription. By performing this assay with BBa_K3806008
as a control, optimal transcription conditions can be found for any ligand. Moreover, this part is provided with the specific prefix and suffix to perform DRIVER, meaning that it can also be used as a positive control during the evolutionary process.

Fig. 3 Urea-PAGE gel to assess the influence of the pH of folate solutions on transcription. Lanes: (Ladder) denatured dsRNA ladder, (1) Negative control: no DNA template. (2) folate solution (pH 14.0), (3) folate solution (pH 9), (4) folate solution (pH 8.5), (5) folate solution (pH 8), (6) folate solution (pH 8) and 10 mM theophylline, and (7) Positive control: without ligand. BBa_K3806008 was used as the DNA template for lanes 2 to 7. The pH of the folate solution was adjusted using NaOH.

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.