User:Brourd/Paper Lab Analyses

Thermodynamic Mimics: Round 1 results summary.

 

Experimental riboswitch design has been stagnant in the cloud, biochemistry game of Eterna, due to the prohibitive cost for the multiple, chemical mapping experiments required, in addition to a very low success rate for designs overall. Recently, Dr. Johan Andreasson, in collaboration with the Das Lab at Stanford University, proposed a method for riboswitch analysis via RNA arrays and fluorescent markers, which is currently an active project that players can take part in. This represents one of many advances for Eterna in riboswitch design, however, there are multiple requirements necessary for the analysis of the riboswitches with Dr. Andreasson’s protocol, as well a lack of single nucleotide resolution, structure mapping measurements.

 

In order to combat the prohibitive costs, as well as gain experimental information about the switching potential of an RNA, the Das Lab created a 6x5 sequence they referred to as a mimic. This mimic is inserted into the 6x5 FMN binding aptamer, internal loop, creating Watson-Crick base pairs that should simulate the binding of FMN to an RNA sequence. Unfortunately, this mimic protocol was not implemented for the design and synthesis of riboswitches in Eterna. However, using experimental data and a reformed mimic protocol, we aim to show that this technique can be used for the successful simulation of riboswitches in vitro.

 

As an overview of the experiment we ran, there were eight targets:

1. Hand and Finger Remade - OFF state: The original hand and finger FMN riboswitch was remade to help combat several issues, this being the OFF state of the RNA.
2. Hand and Finger Remade - Mimic: The origina, Hand and Finger FMN riboswitch was remade to help combat several issues, this being the ON state of the structure, but, with the FMN 6x5 aptamer unlocked, so that players can mutate the loop into one of the many mimics they generated.
3. Simple RNA Switch Remade - OFF State: The original Simple RNA Switch was remade to help combat several issues, this being the OFF state of the RNA.
4. Simple RNA Switch Remade - Mimic: The original Simple RNA Switch was remade to help combat several issues, this being the ON state of the structure, but, with the FMN 6x5 aptamer unlocked, so that players can mutate the loop into one of the mimics they generated.
5. Hair Trigger - Sub 2 (Stable) by Brourd: -2.51 Kcal/mol Mimic Bonus: This Hair Trigger sequence was synthesized in the presence of FMN during the cloud lab switches, ran during round 70 of Eterna. All mimics were pre-generated to have a free energy bonus close to -2.51 kcal/mol, according to either the Vienna 1.8.5 algorithm, or the Vienna 2.1.1 algorithm.
6. Hair Trigger - Sub 2 (Stable) by Brourd: -4.86 Kcal/mol Mimic Bonus: This Hair Trigger sequence was synthesized in the presence of FMN during the cloud lab switches, ran during round 70 of Eterna. All mimics were pre-generated to have a free energy bonus close to -4.86 kcal/mol, according to either the Vienna 1.8.5 algorithm, or the Vienna 2.1.1 algorithm.
7. Bistable 3 - Mod of Eli by mat747: -2.51 Kcal/mol Mimic Bonus: This Bistable 3 sequence was synthesized in the presence of FMN during the cloud lab switches, ran during round 70 of Eterna. All mimics were pre-generated to have a free energy bonus close to -2.51 kcal/mol, according to either the Vienna 1.8.5 algorithm, or the Vienna 2.1.1 algorithm.
8. Bistable 3 - Mod of Eli by mat747: -4.86 Kcal/mol Mimic Bonus: This Bistable 3 sequence was synthesized in the presence of FMN during the cloud lab switches, ran during round 70 of Eterna. All mimics were pre-generated to have a free energy bonus close to -4.86 kcal/mol, according to either the Vienna 1.8.5 algorithm, or the Vienna 2.1.1 algorithm.

 

The hypothesis for this round, was to use an algorithm to procedurally generate FMN mimics for specific free energy values, to try and emulate the free energy bonus a specific concentration of FMN is modeled to have when in the solution with RNA.

 

Specifically, we used the Hair Trigger and Bistable 3 sequences for this.

 

The Hair Trigger ensemble shifted with a concentration 200um FMN, which correlates to a predicted free energy bonus of -2.51 kcal/mol. Therefore, the -2.51 kcal/mol mimics and -4.86 kcal/mol mimics should both indicate a shift in the ensemble.

 

The Bistable 3 ensemble did not shift with a concentration of 200um FMN, which means that it should not switch with the -2.51 kcal/mol mimics. In addition, the ViennaRNA 2.1.1 model predicted that the RNA needed at least -5.8 kcal/mol in order to shift in the ensemble, and therefore, it was hypothesized that the RNA would not successfully switch with the -4.86 kcal/mol mimics either.

 

Results

The results for this indicate with little doubt, that there was a definite correlation between the success of the RNA switch and the free energy bonus. The following graphic has four histograms of the results. On the X axis, is the percentage of switching nucleotides that had a shift in reactivity and on the Y axis is the total number of designs that fall into a specific range.

 

 

 

 

 

 

 

 

For each mimic RNA, if the switching residue is paired in the OFF state and unpaired in the ON state, then the residue gets a full point if the SHAPE reactivity is greater than 0.5.

For each mimic RNA, if the switching residue is paired in the ON state and unpaired in the OFF state, then the residue gets a full point if the SHAPE reactivity is less than 0.5.

For each target, a single residue was removed from the overall switch score, due to data in the control designs, which point towards a consistency in reactivity beteween ON and OFF states.

For now, the Mimic sequence in the 6x5 loop is ignored in scoring, due to the randomness of the sequence and the many structures they may fold into at equilibrium. We are currently looking into ways to improve this.

 

The histograms shown above, group all designs based on the percentage of residues that shift reactivity correctly. Based on this simple analysis, we can make some important observations.

First, none of the mimics for Bistable 3 2.51 kcal/mol had more than 60% of their residues shift in SHAPE reactivity, and the majority of mimic designs had less than 20% of the residues shift reactivity.

Second, 15 out of 33, of the Hair Trigger, 2.51 kcal/mol mimics had 60% or more of their residues shift reactivity. We could most likely infer that this means the mimics had close to a 50% success rate for modeling the presence of FMN with hair trigger -2.51 kcal/mol, but, still not the near 100% success rate we are aiming for.

Third, both the Hair Trigger and Bistable 3 sequences have not been chemically probed in the concetration of FMN necessary to achieve this bonus. On the other hand, the results from both provide some very important details. The most important of these details, is the fact that bistable 3 -4.86 mimics had a switch success rate of less than 50%. Additionally, the distribution of designs would seem to indicate, unlike the Hair Trigger -2.51 mimics, that a mimic was either highly successful, or a total failure. As for Hair Trigger -4.86, almost every mimic switched with more than 60% of residues that shift reactivities, with an overall success rate of 84%.

In conclusion, the proceduarally generated, thermodynamic mimics, were succesful overall, but, were not a 100% simulation of +FMN conditions. However, enough positive evidence has been provided for us to continue with the project, and refine the basic score terms of the fitness algorithm used to generate the mimic sequences. Below are screenshot comparisons of the +FMN data and the Mimics for Hair Trigger -2.51 kcal/mol and Bistable 3 -2.51 kcal/mol.

Hair Trigger

 

 

+FMN

 

 

Mimics

  

 

 

 

It is quite obvious, based on the data we gained, that the ViennaRNA thermodynamic model and nearest neighbor parameters, greatly overestimate the stability of those mimics that have no G-C pairs. Even the thermodynamic mimic with a lonely, G-C base pair, did better than those mimics where the thermodynamic contribution was solely due to A-U and G-U pairs.

 

 

Bistable 3

+FMN

 

Mimics

The Bistable 3 mimics were failures for a number of sequences overall, but, once again, it was not a 100% emulation of +FMN conditions. For example, 5/35 of the synthesized mimics had 40% or more residues shift reactivity. In addition, an interesting point of comparsion here are the 18% and 54% mimics. The main thermodynamic contributions in the sequences are identical, yet, there is a a gap in score that translates to several residues shifting in reactivity. In addition, all of the mimics shown here contain G-C pairs as a part of the generated sequence, and many still failed.

 

Methods

All mimics for these two RNA sequences were procedurally generated using a monte carlo algorithm, that randomly searches through the 4,194,304 different sequences that can potentially be the 6x5 internal loop for the FMN aptamer. By combining the free energy deltas for the ON and OFF state structures of each generated sequence, an approximate free energy bonus is calculated. This is one of the features used to determine the "fitness" of each sequence, in addition to structure distance, and whether or not the mimic misfolds with the RNA sequnce. Mimics that score higher than 0.9 were deemed good enough for use, and that was the what players were advised to use.

 

A list of 80 pre generated mimics were provided for players with each target. 40 of these mimics were generated using the ViennaRNA 1.8.5 parameters, and 40 were generated using the ViennarRNA 2.1.1 parameters. Mimics were mixed to gain unbiased information on both models.

 

Discussion

The chemical mapping data from the first round of the thermodynamic mimics, provides clear evidence of simulating +FMN conditions to a cetain extent. However, various factors, including noncanonical loop interactions and the base pair content of a mimic, will have a significant effect on the stability of the 6x5 motif, and the frequency of the ON and OFF state in the ensemble. The use of custom scoring terms in the Mimic Fitness Algorithm are being implemented, as well as the addition of the partition function of the RNA ensemble. With these additional scoring terms, we aim to better model thermodynamic mimics in round 2 of the project.

 

Furthermore, the use of thermodynamic mimics, whose free energy decreases incrementally, can potentially be used to determine the approximate ligand concentration that causes a shift in conformation for a riboswitch sequence. This pseudo-titration could be especially useful for the robust testing of riboswitch structures, allowing Eterna players to tease out the rules for which sequences switch at which concentrations.

 

A small note. The two novel targets in the project, Hand and Finger Remade, and Simple RNA Switch Remade, both came out with mostly terrible results. High reactivity errors in Hand and Finger Remade, caused a lack of reliable, single-nucleotide resolution data, that is necessary to adequeately score riboswitch sequences. As for Simple RNA switch Remade, it is theorized that a suboptimal structure present in the ensemble caused issues with an uncomplicated switch score.

 

To do list

1. Work on better novel targets for switch design.
2. Update fitness algorithm with custom score terms, and partition function.
3. Get Round 2 going asap.
4. Determine if there are better ways to score switches. After this, post a short summary of the results for the H&F Remade and SRNAS Remade.
5. Find out how difficult it would be to include a basic score function for the mimics used in the 6x5 internal loop.

 

Thanks to players Nando, Meechl, and Tomoe for their hard work and dedication!