I have a question for the organizers. In this challenge we are asked to predict reactivity along the length of the RNA molecule which should correspond to accessibility and base pairing which informs us about the RNA's secondary and tertiary structure. It is known that in nature there are many possible RNA post-translational modifications (PTMs), such as methylation. I am assuming that in the synthesized RNAs being assessed in the DMS / 2A3 assay the RNAs are not modified.
\nMy question is do we know how RNA PTMs might affect secondary structure or reactivity of RNAs? Would it be possible to modify the assay to reflect this additional complexity of RNAs?
", "messages": [ { "id": "2429897", "postDate": "09/08/2023 22:08:49", "content": "I have a question for the organizers. In this challenge we are asked to predict reactivity along the length of the RNA molecule which should correspond to accessibility and base pairing which informs us about the RNA's secondary and tertiary structure. It is known that in nature there are many possible RNA post-translational modifications (PTMs), such as methylation. I am assuming that in the synthesized RNAs being assessed in the DMS / 2A3 assay the RNAs are not modified.
\nMy question is do we know how RNA PTMs might affect secondary structure or reactivity of RNAs? Would it be possible to modify the assay to reflect this additional complexity of RNAs?
", "rawMarkdown": "I have a question for the organizers. In this challenge we are asked to predict reactivity along the length of the RNA molecule which should correspond to accessibility and base pairing which informs us about the RNA's secondary and tertiary structure. It is known that in nature there are many possible RNA post-translational modifications (PTMs), such as methylation. I am assuming that in the synthesized RNAs being assessed in the DMS / 2A3 assay the RNAs are not modified.\n\nMy question is do we know how RNA PTMs might affect secondary structure or reactivity of RNAs? Would it be possible to modify the assay to reflect this additional complexity of RNAs?", "votes": null }, { "id": "2429911", "postDate": "09/08/2023 22:56:49", "content": "The RNA's for this challenge are not chemically modified, except during the reaction step that we use to actually probe the structure.
\nBut if we get good models with this challenge, making RNA's with pseudouridine, m1A, m6A, etc. can be part of a Ribonanza II challenge.
\nOther aspects of RNA behavior that can be captured with slight updates to our experimental protocol include:
\nAll of the above is highly relevant to design of RNA medicines like mRNA vaccines and how RNA's behave out 'in the wild' but not captured in our standard conditions.
\nSo we're very much hoping to see rapid progress on the 'standard' RNA structure prediction challenge through Ribonanza -- we'll be eager to move on to these more elaborate questions after that! ๐
", "rawMarkdown": "The RNA's for this challenge are not chemically modified, except during the reaction step that we use to actually probe the structure. \n\nBut if we get good models with this challenge, making RNA's with pseudouridine, m1A, m6A, etc. can be part of a Ribonanza II challenge.\n\nOther aspects of RNA behavior that can be captured with slight updates to our experimental protocol include:\n\n * Changes in chemical modification patterns at late times in response to initial chemical modifications from 2A3 and DMS.\n * How the RNA folds might change depending on solution condition -- changing temperature, salts, and more.\n * How RNA might fold differently immediately after its transcription, rather than after heating and cooling to help put it into thermodynamically favorable states (which is what we've done here).\n * How RNA folds within living cells vs. in the test tube\n * And much more...\n\nAll of the above is highly relevant to design of RNA medicines like mRNA vaccines and how RNA's behave out 'in the wild' but not captured in our standard conditions.\n\nSo we're very much hoping to see rapid progress on the 'standard' RNA structure prediction challenge through Ribonanza -- we'll be eager to move on to these more elaborate questions after that! ๐", "votes": null }, { "id": "2429923", "postDate": "09/08/2023 23:05:02", "content": "Assumption of Unmodified RNAs: The DMS/2A3 assay is often used to assess RNA secondary structure and reactivity under specific conditions. It is generally assumed that the RNA molecules used in such assays are not post-transcriptionally modified. This simplifies the analysis and interpretation of the data.
\nComplexity of PTMs: RNA PTMs can be highly diverse and complex, with different modifications having varying effects on RNA structure, stability, and function. Some modifications may indeed influence base pairing, accessibility, and overall RNA structure.
\nAssay Modification: While it is possible to modify RNA assays to investigate the effects of specific RNA PTMs, doing so would require careful design and validation of the experimental conditions. Different PTMs may have different impacts, and the assays would need to be tailored accordingly.
\nChallenges in Predicting PTM Effects: Predicting the effects of RNA PTMs on RNA structure is a complex task. It often requires a combination of experimental data and computational modeling to understand how specific modifications influence RNA folding and interactions.
\nResearch Opportunities: Understanding the role of RNA PTMs in RNA structure and function is an active area of research. Researchers are continually exploring how specific modifications impact RNA biology, and this knowledge can be applied to design more sophisticated assays in the future.
", "rawMarkdown": "Assumption of Unmodified RNAs: The DMS/2A3 assay is often used to assess RNA secondary structure and reactivity under specific conditions. It is generally assumed that the RNA molecules used in such assays are not post-transcriptionally modified. This simplifies the analysis and interpretation of the data.\n\nComplexity of PTMs: RNA PTMs can be highly diverse and complex, with different modifications having varying effects on RNA structure, stability, and function. Some modifications may indeed influence base pairing, accessibility, and overall RNA structure.\n\nAssay Modification: While it is possible to modify RNA assays to investigate the effects of specific RNA PTMs, doing so would require careful design and validation of the experimental conditions. Different PTMs may have different impacts, and the assays would need to be tailored accordingly.\n\nChallenges in Predicting PTM Effects: Predicting the effects of RNA PTMs on RNA structure is a complex task. It often requires a combination of experimental data and computational modeling to understand how specific modifications influence RNA folding and interactions.\n\nResearch Opportunities: Understanding the role of RNA PTMs in RNA structure and function is an active area of research. Researchers are continually exploring how specific modifications impact RNA biology, and this knowledge can be applied to design more sophisticated assays in the future.", "votes": null }, { "id": "2429964", "postDate": "09/09/2023 00:13:42", "content": "Another experimental question for you: Is it possible for homomultimer complexes to form? In other words, can two (or more) strands of the same RNA sequence bind with eachother? Or can we be certain that bases only pair with bases in the same strand?
", "rawMarkdown": "Another experimental question for you: Is it possible for homomultimer complexes to form? In other words, can two (or more) strands of the same RNA sequence bind with eachother? Or can we be certain that bases only pair with bases in the same strand?", "votes": null }, { "id": "2430059", "postDate": "09/09/2023 03:42:38", "content": "Given the existence of things like siRNAs I would be very surprised if unconnected strands of the same RNA could not bind to each other!
", "rawMarkdown": "Given the existence of things like siRNAs I would be very surprised if unconnected strands of the same RNA could not bind to each other!", "votes": null }, { "id": "2430060", "postDate": "09/09/2023 03:44:48", "content": "Thank you for the detailed answer! Of course given the complexity of RNA, it's modifications, and interacting partners like RBPs it is curious how closely a 'clean' cell free assay like this one can recapitulate key RNA patterns and phenotypes within a cell.
\nAre there any straightforward validation steps that can be done to probe this potential gap?
", "rawMarkdown": "Thank you for the detailed answer! Of course given the complexity of RNA, it's modifications, and interacting partners like RBPs it is curious how closely a 'clean' cell free assay like this one can recapitulate key RNA patterns and phenotypes within a cell.\n\nAre there any straightforward validation steps that can be done to probe this potential gap?", "votes": null }, { "id": "2432152", "postDate": "09/10/2023 16:03:41", "content": "We cannot be certain that each RNA is forming a structure on it's own. The RNA's are being expressed in libraries, so you can get a sense of what else is in the mix via the sequence lists in the sequence_libraries folder.
For the biochemistry aficionados: The total concentrations of RNA strands is a few micromolar, though any individual RNA sequence will be present at a small fraction of that (1 nanomolar or less).
\nWe do have data showing that when the same RNA molecule is made in different libraries it has similar profiles. This suggests that (1) generally each RNA sequence is not forming structures with molecules of different sequence and (2) structures with the same sequence are less likely, since each RNA sequence's concentration varies across the experiments depending on the number of different sequences and usually stability homomeric structures depends on concentration. However, this general statement may hold true for individual RNA sequences, so perhaps there is extra signal there.
", "rawMarkdown": "We cannot be certain that each RNA is forming a structure on it's own. The RNA's are being expressed in libraries, so you can get a sense of what else is in the mix via the sequence lists in the `sequence_libraries` folder. \n\nFor the biochemistry aficionados: The total concentrations of RNA strands is a few micromolar, though any individual RNA sequence will be present at a small fraction of that (1 nanomolar or less).\n\nWe do have data showing that when the same RNA molecule is made in different libraries it has similar profiles. This suggests that (1) generally each RNA sequence is not forming structures with molecules of different sequence and (2) structures with the same sequence are less likely, since each RNA sequence's concentration varies across the experiments depending on the number of different sequences and usually stability homomeric structures depends on concentration. However, this general statement may hold true for individual RNA sequences, so perhaps there is extra signal there.", "votes": null }, { "id": "2433104", "postDate": "09/11/2023 10:51:48", "content": "Great response, thank you very much
", "rawMarkdown": "Great response, thank you very much", "votes": null } ], "comments": [ { "id": 2429911, "author_name": "rhijudas", "author_url": "", "post_date": "09/08/2023 22:56:49", "content": "The RNA's for this challenge are not chemically modified, except during the reaction step that we use to actually probe the structure.
\nBut if we get good models with this challenge, making RNA's with pseudouridine, m1A, m6A, etc. can be part of a Ribonanza II challenge.
\nOther aspects of RNA behavior that can be captured with slight updates to our experimental protocol include:
\nAll of the above is highly relevant to design of RNA medicines like mRNA vaccines and how RNA's behave out 'in the wild' but not captured in our standard conditions.
\nSo we're very much hoping to see rapid progress on the 'standard' RNA structure prediction challenge through Ribonanza -- we'll be eager to move on to these more elaborate questions after that! ๐
", "votes": null, "replies": [ { "id": 2429964, "author_name": "matthewmasters", "author_url": "", "post_date": "09/09/2023 00:13:42", "content": "Another experimental question for you: Is it possible for homomultimer complexes to form? In other words, can two (or more) strands of the same RNA sequence bind with eachother? Or can we be certain that bases only pair with bases in the same strand?
", "votes": null, "replies": [ { "id": 2430059, "author_name": "zmcxjt", "author_url": "", "post_date": "09/09/2023 03:42:38", "content": "Given the existence of things like siRNAs I would be very surprised if unconnected strands of the same RNA could not bind to each other!
", "votes": null, "replies": [] }, { "id": 2432152, "author_name": "rhijudas", "author_url": "", "post_date": "09/10/2023 16:03:41", "content": "We cannot be certain that each RNA is forming a structure on it's own. The RNA's are being expressed in libraries, so you can get a sense of what else is in the mix via the sequence lists in the sequence_libraries folder.
For the biochemistry aficionados: The total concentrations of RNA strands is a few micromolar, though any individual RNA sequence will be present at a small fraction of that (1 nanomolar or less).
\nWe do have data showing that when the same RNA molecule is made in different libraries it has similar profiles. This suggests that (1) generally each RNA sequence is not forming structures with molecules of different sequence and (2) structures with the same sequence are less likely, since each RNA sequence's concentration varies across the experiments depending on the number of different sequences and usually stability homomeric structures depends on concentration. However, this general statement may hold true for individual RNA sequences, so perhaps there is extra signal there.
", "votes": null, "replies": [ { "id": 2433104, "author_name": "matthewmasters", "author_url": "", "post_date": "09/11/2023 10:51:48", "content": "Great response, thank you very much
", "votes": null, "replies": [] } ] } ] }, { "id": 2430060, "author_name": "zmcxjt", "author_url": "", "post_date": "09/09/2023 03:44:48", "content": "Thank you for the detailed answer! Of course given the complexity of RNA, it's modifications, and interacting partners like RBPs it is curious how closely a 'clean' cell free assay like this one can recapitulate key RNA patterns and phenotypes within a cell.
\nAre there any straightforward validation steps that can be done to probe this potential gap?
", "votes": null, "replies": [] } ] }, { "id": 2429923, "author_name": "mayarmohamedswilam", "author_url": "", "post_date": "09/08/2023 23:05:02", "content": "Assumption of Unmodified RNAs: The DMS/2A3 assay is often used to assess RNA secondary structure and reactivity under specific conditions. It is generally assumed that the RNA molecules used in such assays are not post-transcriptionally modified. This simplifies the analysis and interpretation of the data.
\nComplexity of PTMs: RNA PTMs can be highly diverse and complex, with different modifications having varying effects on RNA structure, stability, and function. Some modifications may indeed influence base pairing, accessibility, and overall RNA structure.
\nAssay Modification: While it is possible to modify RNA assays to investigate the effects of specific RNA PTMs, doing so would require careful design and validation of the experimental conditions. Different PTMs may have different impacts, and the assays would need to be tailored accordingly.
\nChallenges in Predicting PTM Effects: Predicting the effects of RNA PTMs on RNA structure is a complex task. It often requires a combination of experimental data and computational modeling to understand how specific modifications influence RNA folding and interactions.
\nResearch Opportunities: Understanding the role of RNA PTMs in RNA structure and function is an active area of research. Researchers are continually exploring how specific modifications impact RNA biology, and this knowledge can be applied to design more sophisticated assays in the future.
", "votes": null, "replies": [] } ], "raw_markdown_by_id": { "2429897": "I have a question for the organizers. In this challenge we are asked to predict reactivity along the length of the RNA molecule which should correspond to accessibility and base pairing which informs us about the RNA's secondary and tertiary structure. It is known that in nature there are many possible RNA post-translational modifications (PTMs), such as methylation. I am assuming that in the synthesized RNAs being assessed in the DMS / 2A3 assay the RNAs are not modified.\n\nMy question is do we know how RNA PTMs might affect secondary structure or reactivity of RNAs? Would it be possible to modify the assay to reflect this additional complexity of RNAs?", "2429911": "The RNA's for this challenge are not chemically modified, except during the reaction step that we use to actually probe the structure. \n\nBut if we get good models with this challenge, making RNA's with pseudouridine, m1A, m6A, etc. can be part of a Ribonanza II challenge.\n\nOther aspects of RNA behavior that can be captured with slight updates to our experimental protocol include:\n\n * Changes in chemical modification patterns at late times in response to initial chemical modifications from 2A3 and DMS.\n * How the RNA folds might change depending on solution condition -- changing temperature, salts, and more.\n * How RNA might fold differently immediately after its transcription, rather than after heating and cooling to help put it into thermodynamically favorable states (which is what we've done here).\n * How RNA folds within living cells vs. in the test tube\n * And much more...\n\nAll of the above is highly relevant to design of RNA medicines like mRNA vaccines and how RNA's behave out 'in the wild' but not captured in our standard conditions.\n\nSo we're very much hoping to see rapid progress on the 'standard' RNA structure prediction challenge through Ribonanza -- we'll be eager to move on to these more elaborate questions after that! ๐", "2429923": "Assumption of Unmodified RNAs: The DMS/2A3 assay is often used to assess RNA secondary structure and reactivity under specific conditions. It is generally assumed that the RNA molecules used in such assays are not post-transcriptionally modified. This simplifies the analysis and interpretation of the data.\n\nComplexity of PTMs: RNA PTMs can be highly diverse and complex, with different modifications having varying effects on RNA structure, stability, and function. Some modifications may indeed influence base pairing, accessibility, and overall RNA structure.\n\nAssay Modification: While it is possible to modify RNA assays to investigate the effects of specific RNA PTMs, doing so would require careful design and validation of the experimental conditions. Different PTMs may have different impacts, and the assays would need to be tailored accordingly.\n\nChallenges in Predicting PTM Effects: Predicting the effects of RNA PTMs on RNA structure is a complex task. It often requires a combination of experimental data and computational modeling to understand how specific modifications influence RNA folding and interactions.\n\nResearch Opportunities: Understanding the role of RNA PTMs in RNA structure and function is an active area of research. Researchers are continually exploring how specific modifications impact RNA biology, and this knowledge can be applied to design more sophisticated assays in the future.", "2429964": "Another experimental question for you: Is it possible for homomultimer complexes to form? In other words, can two (or more) strands of the same RNA sequence bind with eachother? Or can we be certain that bases only pair with bases in the same strand?", "2430059": "Given the existence of things like siRNAs I would be very surprised if unconnected strands of the same RNA could not bind to each other!", "2430060": "Thank you for the detailed answer! Of course given the complexity of RNA, it's modifications, and interacting partners like RBPs it is curious how closely a 'clean' cell free assay like this one can recapitulate key RNA patterns and phenotypes within a cell.\n\nAre there any straightforward validation steps that can be done to probe this potential gap?", "2432152": "We cannot be certain that each RNA is forming a structure on it's own. The RNA's are being expressed in libraries, so you can get a sense of what else is in the mix via the sequence lists in the `sequence_libraries` folder. \n\nFor the biochemistry aficionados: The total concentrations of RNA strands is a few micromolar, though any individual RNA sequence will be present at a small fraction of that (1 nanomolar or less).\n\nWe do have data showing that when the same RNA molecule is made in different libraries it has similar profiles. This suggests that (1) generally each RNA sequence is not forming structures with molecules of different sequence and (2) structures with the same sequence are less likely, since each RNA sequence's concentration varies across the experiments depending on the number of different sequences and usually stability homomeric structures depends on concentration. However, this general statement may hold true for individual RNA sequences, so perhaps there is extra signal there.", "2433104": "Great response, thank you very much" }, "source": "meta" }