Modelling RNA Aptamer Structures
Our project utilized RNA aptamers as biosensors. We aimed to test these biosensors using RNA Mango as a fluorescent reporter as it would bind to thiazole orange and glow brightly if correctly folded. To understand the interplay between the various RNA conformations possible of our biosensors, we chose to model the biosensors with and without the RNA Mango attached. We used RNAfold WebServer and RNAstructure to predict the secondary structures of our biosensors. We then used those predictions to create a 3D model of the RNA with RNAComposerRNA Mango StructureBBa K4351010
This structure is an aptamer used in our first stages of proof of concept, allowing us to observe the function of our Glucose, Insulin and Theophylline aptamers. RNA mango alone lacks a strong intrinsic fluorescence, but in the presence of thiazole orange increases its fluorescence up to 1,100 fold. RNA Mango forms a very interesting structure, a G-quadruplex, where the repeating G bases stack on top of each other (Autour et al., 2018; Popenda et al., 2019). These G tetrads form the basis for the binding platform for the fluorophore. Several crystal structures have been reported for slightly different RNA Mango sequences, but they all form this same overall structure (Figure 1).
RNA Mango Models
Our testing in the lab used the RNA mango aptamer to visualize the function and potential progress of the formation and bonding of the other aptamers with their corresponding target molecule. Interestingly, the modelling software did not consider the formation of a G-quadruplex structure. Instead a large open circle is shown to form (Figure 2). Even when we bias the prediction software using the crystal structure as a starting point, the RNA does not show the well-organized conformation of stacking G tetrads (Figure 3). The secondary structures were obtained through two different software to compare structures and free energy values.
Glucose Aptamer 1BBa K4351000
We predicted that glucose would be able to bind to and stabilize a stem loop structure. The listed free energy measurements are the amounts of energy required to form/break the nucleotide bonds of the structures. This is important when considering the probability of the aptamers forming in the sequence in the presence of the target molecules. The larger the negative value is, the more stable the structure will be once formed. A higher value is advantageous as it communicates a stable structure once formed, but also conversely requires more energy to form. Both software tools show similar stem loop structures with free energy values around -7 to -8 kcal/mol (Figure 4 and Figure 5).
Glucose Aptamer 1 with RNA MangoBBa K4351018
This structure is one of the aptamers that we were able to test in the lab (https://2022.igem.wiki/lethbridge-hs/proof-of-concept.html) The software tools again did not predict the G-quadruplex formation of RNA Mango and instead show long extended stem loops (Figure 6). This structure is predicted to have free energies between -21 and -23 kcal/mol, indicating more stable conformation than either the Glucose aptamer or RNA Mango aptamers by themselves. This more negative free energy value could indicate that transitioning from this structure to one that forms the glucose binding stem loop and the G-quadruplex for fluorophore binding would be unfavourable. The 3D structure prediction when RNA Mango is forced to form a G-quadruplex structure, demonstrates how we hope the RNA conformation is forming in the presence of ligand (Figure 7)
Glucose ScrambleBBa K4351012
This sequence serves as a negative control of our project, being a sequence that should not result in the binding of glucose. The MFE structure and the Centroid structures differs in their stacking and hairpin loops, or lack thereof. The MFE structure has two hairpin loops and stacking in the middle. The Centroid Structure only consists of one internal loop (Figure 8).
Glucose Scramble with RNA MangoBBa K4351020
This structure is one of the aptamers that we were able to test in the lab (https://2022.igem.wiki/lethbridge-hs/proof-of-concept.html). Once again, the software tools did not predict the G-quadruplex formation for RNA Mango and instead the structure gives a stem with several internal loops (Figure 9). Both software algorithms calculated similar free energy values for the structures. Interestingly, the structure looks like the predicted single scramble and RNA Mango sequences (Figures 2 and 8). When we examine the 3D structures we can see that the RNA Mango forms a very similar structure as predicted previously for RNA Mango alone. The extended internal loop where the G-quadruplex is supposed to form is present. When we bias the software to form the G-quadruplex structure, it forms a much more compact conformation (Figure 10).
Theophylline AptamerBBa K4351013
This sequence was to serve as a positive control, as it has been shown previously to bind to its ligand (theophylline) with tight affinity (Rankin et al., 2006). Here, the MFE structure and Centroid structure are identical to each other (Figure 11). Both software tools predicted a short stem with a four-nucleotide hairpin loop, a small bulge loop and a internal loop.
Theophylline Aptamer with RNA MangoBBa K4351021
This structure is one of the aptamers that we were able to test in the lab (https://2022.igem.wiki/lethbridge-hs/proof-of-concept.html). The theophylline with RNA Mango RNA is predicted to have several internal loops and base pairing between part of the stem of RNA Mango and the theophylline stem (Figure 12). These structures have free energies of -19 to -21 kcal/mol and are more stable than the theophylline aptamer by itself. The predicted 3D structure shows two extended stem loops with base pairing between the RNA Mango sequence and the theophylline sequence. In the biased G-quadruplex structure, you no longer see this base pairing and instead see the theophylline stem loop and the RNA Mango structures forming independently (Figure 13).
Conclusions and Next Steps:
The overall larger free energy values for the aptamers when combined with RNA Mango was not surprizing as the longer RNA sequences offer more base pairing opportunities but it may allow us to better understand the energy required to move between different RNA conformations. Since the predictions algorithms were unable to predict the formation of the RNA Mango G-quadruplex, more information will be required to determine the effect of the formation of this structure on the overall stability of the RNA.
Modelling the RNA aptamers has been very helpful to our team in understanding how RNA sequences is linked to secondary and tertiary structures and how those sequences have an impact on how these biomolecules function.
We would like to use our models to help us design our next RNA aptamers for insulin detection.
References:
[1] Autour, Alexis & Jeng, Sunny & Cawte, Adam & Abdolahzadeh, Amir & Galli, Angela & Panchapakesan, Shanker & Rueda, David & Ryckelynck, Michael & Unrau, Peter. (2018). Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells. Nature Communications. 9. 10.1038/s41467-018-02993-8.
[2] Popenda, Mariusz & Miśkiewicz, Joanna & Sarzynska, Joanna & Zok, Tomasz & Szachniuk, Marta. (2019). Topology-based classification of tetrads and quadruplex structures. Bioinformatics (Oxford, England). 36. 10.1093/bioinformatics/btz738.
[3] Rankin, C. J.; Fuller, E. N.; Hamor, K. H.; Gabarra, S. A.; Shields, T. P. (2006). A Simple Fluorescent Biosensor for Theophylline Based on its RNA Aptamer. Nucleosides, Nucleotides and Nucleic Acids, 25(12), 1407–1424. doi:10.1080/15257770600919084
Glucose Aptamer 1 with RNA MangoBBa K4351018
This structure is one of the aptamers that we were able to test in the lab (https://2022.igem.wiki/lethbridge-hs/proof-of-concept.html) The software tools again did not predict the G-quadruplex formation of RNA Mango and instead show long extended stem loops (Figure 6). This structure is predicted to have free energies between -21 and -23 kcal/mol, indicating more stable conformation than either the Glucose aptamer or RNA Mango aptamers by themselves. This more negative free energy value could indicate that transitioning from this structure to one that forms the glucose binding stem loop and the G-quadruplex for fluorophore binding would be unfavourable. The 3D structure prediction when RNA Mango is forced to form a G-quadruplex structure, demonstrates how we hope the RNA conformation is forming in the presence of ligand (Figure 7)
Glucose ScrambleBBa K4351012
This sequence serves as a negative control of our project, being a sequence that should not result in the binding of glucose. The MFE structure and the Centroid structures differs in their stacking and hairpin loops, or lack thereof. The MFE structure has two hairpin loops and stacking in the middle. The Centroid Structure only consists of one internal loop (Figure 8).
Glucose Scramble with RNA MangoBBa K4351020
This structure is one of the aptamers that we were able to test in the lab (https://2022.igem.wiki/lethbridge-hs/proof-of-concept.html). Once again, the software tools did not predict the G-quadruplex formation for RNA Mango and instead the structure gives a stem with several internal loops (Figure 9). Both software algorithms calculated similar free energy values for the structures. Interestingly, the structure looks like the predicted single scramble and RNA Mango sequences (Figures 2 and 8). When we examine the 3D structures we can see that the RNA Mango forms a very similar structure as predicted previously for RNA Mango alone. The extended internal loop where the G-quadruplex is supposed to form is present. When we bias the software to form the G-quadruplex structure, it forms a much more compact conformation (Figure 10).
Theophylline AptamerBBa K4351013
This sequence was to serve as a positive control, as it has been shown previously to bind to its ligand (theophylline) with tight affinity (Rankin et al., 2006). Here, the MFE structure and Centroid structure are identical to each other (Figure 11). Both software tools predicted a short stem with a four-nucleotide hairpin loop, a small bulge loop and a internal loop.
Theophylline Aptamer with RNA MangoBBa K4351021
This structure is one of the aptamers that we were able to test in the lab (https://2022.igem.wiki/lethbridge-hs/proof-of-concept.html). The theophylline with RNA Mango RNA is predicted to have several internal loops and base pairing between part of the stem of RNA Mango and the theophylline stem (Figure 12). These structures have free energies of -19 to -21 kcal/mol and are more stable than the theophylline aptamer by itself. The predicted 3D structure shows two extended stem loops with base pairing between the RNA Mango sequence and the theophylline sequence. In the biased G-quadruplex structure, you no longer see this base pairing and instead see the theophylline stem loop and the RNA Mango structures forming independently (Figure 13).
Conclusions and Next Steps:
The overall larger free energy values for the aptamers when combined with RNA Mango was not surprizing as the longer RNA sequences offer more base pairing opportunities but it may allow us to better understand the energy required to move between different RNA conformations. Since the predictions algorithms were unable to predict the formation of the RNA Mango G-quadruplex, more information will be required to determine the effect of the formation of this structure on the overall stability of the RNA.
Modelling the RNA aptamers has been very helpful to our team in understanding how RNA sequences is linked to secondary and tertiary structures and how those sequences have an impact on how these biomolecules function.
We would like to use our models to help us design our next RNA aptamers for insulin detection.
References:
[1] Autour, Alexis & Jeng, Sunny & Cawte, Adam & Abdolahzadeh, Amir & Galli, Angela & Panchapakesan, Shanker & Rueda, David & Ryckelynck, Michael & Unrau, Peter. (2018). Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells. Nature Communications. 9. 10.1038/s41467-018-02993-8.
[2] Popenda, Mariusz & Miśkiewicz, Joanna & Sarzynska, Joanna & Zok, Tomasz & Szachniuk, Marta. (2019). Topology-based classification of tetrads and quadruplex structures. Bioinformatics (Oxford, England). 36. 10.1093/bioinformatics/btz738.
[3] Rankin, C. J.; Fuller, E. N.; Hamor, K. H.; Gabarra, S. A.; Shields, T. P. (2006). A Simple Fluorescent Biosensor for Theophylline Based on its RNA Aptamer. Nucleosides, Nucleotides and Nucleic Acids, 25(12), 1407–1424. doi:10.1080/15257770600919084
Glucose Scramble with RNA MangoBBa K4351020
This structure is one of the aptamers that we were able to test in the lab (https://2022.igem.wiki/lethbridge-hs/proof-of-concept.html). Once again, the software tools did not predict the G-quadruplex formation for RNA Mango and instead the structure gives a stem with several internal loops (Figure 9). Both software algorithms calculated similar free energy values for the structures. Interestingly, the structure looks like the predicted single scramble and RNA Mango sequences (Figures 2 and 8). When we examine the 3D structures we can see that the RNA Mango forms a very similar structure as predicted previously for RNA Mango alone. The extended internal loop where the G-quadruplex is supposed to form is present. When we bias the software to form the G-quadruplex structure, it forms a much more compact conformation (Figure 10).
Theophylline AptamerBBa K4351013
This sequence was to serve as a positive control, as it has been shown previously to bind to its ligand (theophylline) with tight affinity (Rankin et al., 2006). Here, the MFE structure and Centroid structure are identical to each other (Figure 11). Both software tools predicted a short stem with a four-nucleotide hairpin loop, a small bulge loop and a internal loop.
Theophylline Aptamer with RNA MangoBBa K4351021
This structure is one of the aptamers that we were able to test in the lab (https://2022.igem.wiki/lethbridge-hs/proof-of-concept.html). The theophylline with RNA Mango RNA is predicted to have several internal loops and base pairing between part of the stem of RNA Mango and the theophylline stem (Figure 12). These structures have free energies of -19 to -21 kcal/mol and are more stable than the theophylline aptamer by itself. The predicted 3D structure shows two extended stem loops with base pairing between the RNA Mango sequence and the theophylline sequence. In the biased G-quadruplex structure, you no longer see this base pairing and instead see the theophylline stem loop and the RNA Mango structures forming independently (Figure 13).
Conclusions and Next Steps:
The overall larger free energy values for the aptamers when combined with RNA Mango was not surprizing as the longer RNA sequences offer more base pairing opportunities but it may allow us to better understand the energy required to move between different RNA conformations. Since the predictions algorithms were unable to predict the formation of the RNA Mango G-quadruplex, more information will be required to determine the effect of the formation of this structure on the overall stability of the RNA.
Modelling the RNA aptamers has been very helpful to our team in understanding how RNA sequences is linked to secondary and tertiary structures and how those sequences have an impact on how these biomolecules function.
We would like to use our models to help us design our next RNA aptamers for insulin detection.
References:
[1] Autour, Alexis & Jeng, Sunny & Cawte, Adam & Abdolahzadeh, Amir & Galli, Angela & Panchapakesan, Shanker & Rueda, David & Ryckelynck, Michael & Unrau, Peter. (2018). Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells. Nature Communications. 9. 10.1038/s41467-018-02993-8.
[2] Popenda, Mariusz & Miśkiewicz, Joanna & Sarzynska, Joanna & Zok, Tomasz & Szachniuk, Marta. (2019). Topology-based classification of tetrads and quadruplex structures. Bioinformatics (Oxford, England). 36. 10.1093/bioinformatics/btz738.
[3] Rankin, C. J.; Fuller, E. N.; Hamor, K. H.; Gabarra, S. A.; Shields, T. P. (2006). A Simple Fluorescent Biosensor for Theophylline Based on its RNA Aptamer. Nucleosides, Nucleotides and Nucleic Acids, 25(12), 1407–1424. doi:10.1080/15257770600919084