Part:BBa_K3806008

Theophylline-binding aptazyme

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]. Aptazymes can be used in large number scenarios, ranging from disease diagnosis, prognosis, or treatment to detecting small pollutants in the environment. They 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) performed a computational study of the theophylline-binding aptazyme, (ii) validated the cleavage activity of this aptazyme using a Urea-PAGE gel following a co-transcriptional cleavage assay, (iii) showed the value of BBa_K3806008 when used as a positive control in the setup and course of DRIVER experiments, and (iv) 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

Molecular dynamics of the theophylline-binding aptazyme

In order to get a better understanding of the theophylline-binding aptazyme, a Nanoscale Molecular Dynamics (NAMD) computational analysis was performed. Molecular dynamics (MD) is a valuable and sophisticated computational tool to probe the dynamic evolution of molecular systems, providing a time-dependent picture that emerges from interatomic interactions.

MD simulations were performed with NAMD [3] over simulation times of 50 ns using the CHARMM force field. The initial structure of the binding-theophylline aptazyme was predicted with iFoldRNA (https://dokhlab.med.psu.edu/ifoldrna/) and prepared for the MD simulation using the PDB Reader service of CHARMM-GUI (http://www.charmm-gui.org/) [4]. Theophylline was not considered in the analysis. Periodic solvation boxes were constructed with 14 Å spacing and water molecules according to the TIP3P model [5]. Sodium and chloride ions were added to counter the total charges of the RNA molecule setting a 0.150 M salt concentration. The particle-mesh Ewald summation method [6] was used for long-range electrostatics and a 10 Å cutoff was set for short-range non-bonded interactions. Initial geometries were first minimised at 3,000 conjugate-gradient steps, water was then equilibrated at 298 K and 1 atm for 100 ps at 2 fs time steps, and production runs were then performed for 50 ns at 2 fs time steps (25 million steps per calculation) in the NPT ensemble at 1 atm and 298 K. Langevin dynamics for T control and the Nosé-Hoover Langevin piston method for P control were employed. NAMD output was stored every 12,500 steps, giving trajectories composed of 2,000 frames that were processed and analysed with VMD 1.9 [7] and PyMOL 1.4 (pymol.org).

Structures after 0, 30, and 50 ns of simulation in the abscense of theophylline were compared (Fig. 2). From this figure, it can be seen that the helix-like structure in the apex of the aptazyme (yellow square) is lost to enable bending of the aptazyme in order to approximate the nucleotides involved in the auto-catalytic reaction to the cleavage point (pink arrow). This behaviour of the aptazyme is expected when theophylline is not present, corresponding to the modeling conditions of this study. Although not modeled here, binding of the ligand to specific sequences within the aptazyme loops is expected to hinder this process.

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Fig. 2 3D structure of the theophylline-binding aptazyme (BBa_K3806008) after 0, 30 and 50 ns of MD simulation. The cleavage site is indicated with a pink arrow. The aptazyme bends to approximate the nucleotides involved in the auto-catalytic reaction to the cleavage point.

Cleavage characterization of the theophylline-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. 3)

Fig. 3 Urea-PAGE gel for characterization of theophylline-binding aptazyme cleavage (BBa_K3806008) fractions following a co-transcriptional cleavage assay.. Lanes: (Ladder) denatured dsRNA ladder, (1) Negative control: no DNA template. (2) no DNA template (without T7 RNA polymerase added to the transcription reaction), transcription of BBa_K3806008 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. 3, 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, using BBa_K3806008 as the DNA template for transcription (Fig. 4). 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 possible as well. 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 (W-prefix) and suffix (X-suffix) to perform DRIVER, meaning that it can also be used as a positive control during the evolutionary process.

Fig. 4 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.

Aptazyme-regulated LacZ expression

BBa_K3806008 can be incorporated in a genetic circuit to have a ligand-dependent regulated gene expression. For instance, the design of the BBa_K3806014 part was based on BBa_K3806008. The aptazyme without the DRIVER prefix and suffix is enclosed within the ribosomal binding site (RBS) and an antisense strand complementary to the RBS (anti-RBS), and fused to a lacZ gene. Due to the secondary structure of the aptazyme, the anti-RBS is able to approach and sequester the RBS. When the cleavage of the aptazyme occurs, the RBS is liberated from the anti-RBS, allowing for translation initiation. For a colorimetric characterization of BBa_K3806014 containing the theophylline-binding aptazyme, this part was expressed in PURExpress using CPRG as a substrate. As expected, a decrease in expression can be seen when theophylline is added to the reaction as binding of theophylline preserves the structure of the aptazyme resulting in sequestering of the RBS (Fig. 5).

Fig. 5 Image of BBa_K3806014 expression in PURExpress using CPRG as substrate. Samples: (1) without BBa_K3806014 and 0 mM theophylline, yellow; (2) without BBa_K3806014 and 6 mM theophylline, yellow; (3) with BBa_K3806014 and 0 mM theophylline, red; and (4) BBa_K3806014 and 6 mM theophylline, orange. BBa_K3806014 was added at a final concentration of 5 nM. CPRG was used as a substrate to a final concentration of 0.6 mg/ml.

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.
3. [3] Phillips, J.C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R.D., Kalé, L., & Schulten, K. (2005). Scalable molecular dynamics with NAMD. Journal of Computational Chemistry, 26(16), 1781–1802.
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6. [6] Darden, T., York, D., & Pedersen, L. (1993). Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems. Journal of Chemical Physics, 98, 10089-10092.
7. [7] Humphrey, W., Dalke, A., & Schulten, K. (1996). VMD: visual molecular dynamics. Journal of Molecular Graphics, 14(1), 33–28. https://doi.org/10.1016/0263-7855(96)00018-5