Organization

The experimental work of our project aims to complete the various cycles described in the Engineering section. In the table below, each wet lab branch is associated with the engineering cycle it seeks to complete and provide experimental validation for.

Engineering	Wet Lab
Branch: SCR-D structure prediction Cycle 2: Identification of key structural elements - Complete iteration 3	Branch 1: SCR-D element
Branch: Creation of an aptamer-controlled SCR Cycle 1: SCR element - Complete iteration 3	Branch 2: SECIS Branch 5: SECIS Cell-free
Branch: Creation of an aptamer-controlled SCR Cycle 2: Aptamer - Complete iteration 2	Branch 3: Aptamer Branch 4: Aptamer Cell-free
Branch: Creation of an aptamer-controlled SCR Cycle 3: Riboswitch - Complete iteration 1	Branch 6: Switch

A detailed explanation of the workflow Engineering → Wet Lab can be found in the Model Page.

Experimental workflow

General scheme of experimental workflow

In the laboratory, we mainly carried out two types of assays: cell-based assays and cell-free system assays. Both assays share a common preliminary part.

Common part

The constructs arrived from the supplier in plasmid form. In order to amplify the plasmids, we performed a standard amplification procedure using E. coli DH5α:

Transformation: We carried out a bacterial transformation by heat shock of E. coli DH5α with the plasmids of interest, and plated the bacteria.
Incubation: The following day, we collected colonies from the plates and incubated them in tubes with LB medium.
Plasmid purification: One day later, we purified the plasmids of interest, obtaining the desired concentration.
Verification: Finally, to verify the efficiency of the procedure, we measured the concentration of the purified plasmids using a Nanodrop.

All plasmids underwent this process before being used in the assays.

Cell-based assays in HEK293T

Starting from the purified plasmids, we performed the assays in HEK293T cells, with plasmids of SCR-D and SECIS. A complete assay takes one week, so we always began on Monday and finished over the weekend. The process is as follows:

Weekly Experimental Timeline

From cell seeding to data analysis - a complete 7-day protocol

Monday

Cell Seeding

We seeded the cells at the desired density in 48-well plates. The cells were then incubated for 24 hours.

Tuesday

Transfection

We transfected the plasmids of interest (SCR-D and SECIS) into the cells using a lipofectamine-based system, followed by a 24-hour incubation.

Wednesday

Treatment

After preparing selenium and theophylline solutions, we treated the cells with selenium and/or theophylline and incubated them for 48 hours.

Thursday

Observation

We observed the cells under an optical microscope, solely to check that the process was proceeding correctly and that cell growth was taking place as expected.

Friday

Luciferase Assay

We performed cell lysis and dual-luciferase assay 💡(Dual-Luciferase® Reporter Assay System from Promega®, catalog #E1910), obtaining raw data on gene expression.

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Weekend

Data Analysis & Presentation

We dedicated this time to plotting and analyzing the results in RStudio, and prepared a presentation of the findings and conclusions to share with our PI and instructors.

This procedure shows variations across the different rounds carried out, since we have conducted an optimization process of the various variables and time points that influence the experiment. For more details about this process, please refer to the Pivotal changes and milestones Page.

Cell-free system assays

Starting from the purified plasmids, we performed the cell-free system assays (TnT® Coupled Reticulocyte Lysate Systems from Promega®, catalog #L1170), but in this case only SECIS plasmids and SECIS-related plasmids were tested. A complete assay takes one or two days. The process is as follows:

Starting Material: The starting point can be either a purified plasmid or a PCR product. We incorporated Paul Copeland’s plasmid in purified plasmid form. However, we performed a PCR on the SECIS plasmids to incorporate our construct as a PCR product, since in this form it was more stable. Therefore, the first step was to run a PCR and an agarose gel electrophoresis.
Assay Setup: Once we had the PCR product, we proceeded with the cell-free system assay. The different samples were treated with selenium and/or theophylline, which were added prior to the incubation period during which transcription and translation occurred simultaneously.
Measurement: Gene expression was measured through a dual-luciferase assay. Finally, we plotted and analyzed the results in RStudio and prepared a presentation to share with our PI and instructors.

Other Procedures

Split: This routine procedure in the team is used to replace the medium of HEK293T cells with trypsin, thereby ensuring their survival over time.
Toxicity assay: This procedure involves seeding cells at different densities and treating them with increasing concentrations of theophylline, in order to establish toxicity thresholds.
Sanger sequencing verification: To confirm the identity and integrity of the purified constructs, plasmid samples are sent for Sanger sequencing. The results are compared to the expected construct sequences to verify that they match the intended design.

More information about lab procedures and protocols can be found at the Notebook section.

Constructs used in the lab

The constructs used in our laboratory are divided into two main branches, according to the SCR with which they are associated. Thus, we have constructs that were used for the SCR-D experiments and constructs that were used for the SECIS experiments.

Classification of the constructs that we used in the lab

SCR-D

A: Normal SCR-D (TGA)

This construct is the complete SCR-D along with the STOP codon on which it must act to produce readthrough, allowing the ribosome to bypass it. This way, Firefly luciferase will be expressed.

Normal SCR-D (TGA) construct and predicted structure

B: SCR-D controls

1- CGA: This construct has the first nucleotide of the STOP codon mutated from T to C, so the codon is no longer a STOP codon but instead encodes an arginine. In other words, each reading of the construct’s mRNA gives rise to both luciferase proteins, Renilla and Firefly. This serves to normalize the activity of the two luciferases and to generate cleaner and more reliable graphs.

CGA construct, a control for normalizing the activity of both luciferases

2- TAAA: This construct serves as a negative control for the Dual-Luciferase Assay. In this case, the STOP codon was modified by changing its central nucleotide (G → A) and adding one extra nucleotide immediately after it, introducing a frameshift. As a result, even if the ribosome continues translation beyond this point, the downstream Firefly luciferase gene will be out of frame and cannot be correctly translated. Therefore, no Firefly signal should be detected. This control ensures that any measured Firefly activity in experimental constructs truly results from readthrough events rather than from frameshifting or reinitiation.

TAAA construct, useful for ruling out reinitiation and other modes of Firefly luciferase expression that are independent of SECIS-mediated readthrough.

3- TGA postATG mutated: This construct was designed to control for ribosomal reinitiation. In the natural SCR-D sequence, there is an internal ATG start codon located downstream of the stop codon. This ATG could allow the ribosome to detach and then reinitiate translation, leading to Firefly luciferase expression that is not due to true readthrough. To prevent this, the internal ATG was mutated in a way that preserved the SCR-D RNA structure while eliminating the reinitiation site. As a result, any Firefly luciferase produced from this construct must come exclusively from readthrough events. By comparing this construct to the normal SCR-D (TGA) construct, we can quantify and subtract the contribution of reinitiation.

TGA postATG mutated construct, useful for ruling out reinitiation, a mode of Firefly luciferase expression that is independent of SECIS-mediated readthrough.

C: SCR-D parts

1- Only pre-diapin: This construct contains only the Pre-diapin region of SCR-D, and it is used to test whether this isolated part is capable of producing readthrough on its own. It allows us to quantify the percentage contribution of Pre-diapin to readthrough, if it is indeed able to induce it.

SCR-D construct containing only the pre-diapin element

2- No hypopin: This construct contains all parts of SCR-D except for the Hypopin. It is used to determine whether this region is essential for readthrough to occur, or if SCR-D can bypass the STOP codon without it. It allows us to quantify the percentage contribution of Hypopin to readthrough.

SCR-D construct not containing hypopin element

3- Mutated Diapin: This construct is nearly identical to the normal SCR-D (TGA) element, except that the nucleotide sequence of the Diapin region was altered while maintaining its predicted base pairing and secondary structure. It was designed to determine whether the Diapin’s specific sequence is essential for promoting readthrough, or whether its structural configuration alone is sufficient for SCR-D function. This distinction is particularly important for our project, as we are exploring structure-dependent mechanisms to build a riboswitch.

SCR-D construct containing a mutated diapin element

SECIS

A: Controls

1- Activity normalization: The construct used to normalize luciferase activity in the SECIS experiments was the same as the one used in SCR-D. Since both SCR-D and SECIS employ the same luciferases to measure gene expression, we were able to use the SCR-D luciferase normalization construct for normalization in the SECIS experiments.

CGA construct, a control for normalizing the activity of both luciferases

2- Negative control / Bad SECIS: This construct does not contain any element that enables readthrough, and is therefore used to determine the amount of Firefly luciferase produced as a result of spontaneous ribosomal readthrough, experimental background, reinitiation, or other possible experimental artifacts. Thus, in constructs with SECIS, we can determine what percentage of Firefly luciferase corresponds to this background noise and what percentage actually corresponds to the readthrough driven by SECIS.

Bad SECIS allows us to rule out Firefly luciferase expression that is independent of SECIS-mediated readthrough.

3- SECIS control: This is the construct we used to verify that the SCR element of our riboswitch, namely SECIS, functions on its own. This construct is very important because, before testing our riboswitch, it allowed us to confirm that the SECIS element works and is in the proper conditions to carry out readthrough.

SECIS control confirmed that SECIS system works in the experimental conditions we are exposing it to

4- Aptamer control: This construct allows us to verify the aptamer’s structural responsiveness to the ligand under our experimental conditions before testing it within the riboswitch context. In this design, the presence or absence of theophylline influences the formation of a pseudoknot structure that can induce ribosomal frameshifting. If no theophylline is present, the pseudoknot that induces ribosomal frameshift does not fold correctly. Conversely, when theophylline is added, the pseudoknot is released, allowing the ribosome to undergo frameshift and avoid a STOP codon, placing Firefly luciferase in the correct ORF and resulting in its expression. This sequence and design is extracted from Anzalone et al., Reprogramming eukaryotic translation with ligand-responsive synthetic RNAb switches, Nature Methods (2016). https://doi.org/10.1038/nmeth.3807

Dr Anzalone’s construct is crucial to make sure the aptamer is working in the experimental conditions we are exposing it to.

B: Switches

The following constructs represent different designs of the SECIS-based riboswitch, coupled with the theophylline aptamer. In their names, the letter L stands for “Linker,” and the accompanying number indicates the number of nucleotides inserted between the SECIS element and the aptamer. The L5 construct has a slightly modified version of the SECIS sequence.
In the absence of theophylline, the aptamer base-pairs with part of the SECIS sequence, keeping the two regions bound together and preventing SECIS from folding into its natural structure. The aptamer also base-pairs with the linker when there is no theophylline. When theophylline binds to the aptamer, this interaction is disrupted, changing the conformation of the SECIS region and therefore altering Firefly luciferase expression and acting as a riboswitch for readthrough rate.

1- L5: Structure of Linker 5 construct.

Structure of Linker 5 construct

2- L7: Structure of Linker 7 construct.

Structure of Linker 7 construct

3- L8: Structure of Linker 8 construct.

Structure of Linker 8 construct

4- L9: Structure of Linker 9 construct.

Structure of Linker 9 construct

It is important to consider that these four riboswitches were designed using our model and the TADPOLE software. These two tools operate based on structural predictions provided by RNAfold and focus specifically on the energetic requirements that allow the aptamer to fluctuate between two states: bound to theophylline or unbound. The linkers were designed based on an ON-ON switch structural prediction with RNAfold. This means that a functional (non-disrupted) SECIS element is expected in the presence of theophylline, as illustrated in the riboswitch structures.
However, RNAfold has known limitations that prevent its structural predictions from being fully accurate, and these limitations appear to affect the non-canonical interactions within the functional core of SECIS. Therefore, although the in silico prediction suggested an ON-ON switch, an OFF-ON switch may be observed experimentally.

C: Dr. Copeland’s plasmid

This plasmid was used in the cell-free assays as a supplement for SECIS. The SBP2 protein is essential for SECIS to induce readthrough, so its presence is required to achieve higher readthrough efficiency.

Simplified version of Dr. Paul Copeland’s SBP2 gene context.

Experimental Overview