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Eterna Project Information Project Information Overview, History, and Objectives Prepared By: Jonathan Romano Last Revised: 04/15/2021 Revision History Rev. Date Author Revision Description 1 02/28/2020 Jonathan Romano Initial copy 2 02/29/2020 Rhiju Das Copy improvements, historical clarification, improvements to platform development goals 3 03/17/2020 Leonard Copy improvements, move content to Oppenheimer software appendix, additional community section, project note for switch design 4 03/18/2020 Jonathan Romano Copy improvements, add note of upcoming COVID-19 challenge 5 04/14/2021 Sharif Ezzat Copy updates, removed value proposition, updated COVID-19 challenge information Acknowledgements This document is based on prior work by and discussions with a number of individuals affiliated with the Eterna project, including Rhiju Das, Benjamin Keep, Do Soon Kim, Leonard Oppenheimer, Jennifer Pearl, and Boris Rudolfs. 1 Table of Contents Revision History 1 Acknowledgements 1 Table of Contents 2 Introduction 3 Purpose 3 Intended Audience 3 Eterna Project Information 3 Overview 3 Description 3 Notable Achievements 4 Projects 4 Papers 4 Press Coverage 5 History 5 Origins 5 Science 5 Software 6 Players/Community 7 Future Objectives 8 Consortium/Commons Organization 8 Appendix 1: Eterna Software 9 Eterna’s Software Development History 9 Eterna’s Future Software Development 10 2 Introduction Purpose This document provides information about the Eterna project, how it has developed over time, and plans for how it might be maintained and expanded in the future. Intended Audience This background document helps educate Eterna staff, media, volunteers, and potential and actual commercial, academic, and funding partners. Eterna Project Information Overview Description Eterna is first and foremost a citizen science game, supporting research in basic and applied research to predict and design synthetic ribonucleic acid (RNA) structures. RNA, the extremely powerful molecule at the heart of many cellular processes, acts as the operating system of life based on how it folds into complex shapes. RNA research has the potential to lead to breakthroughs in diagnosing and treating dangerous and wide-spread diseases. RNA research may also lead to the development of new technologies, outside of medicine, that are currently impossible through conventional means. However, scientists don’t fully understand RNA’s behavior. Creating synthetic designs to carry out a specific function and predicting how well they will perform when created, is extremely difficult - but the diverse community of Eterna players, representing a wide variety of ages, locations, and backgrounds, have tackled this challenge head on, and have done so with great success. The best players still remain better at designing synthetic RNA and solving RNA secondary structures than the best computer algorithms. Through the abstraction of a puzzle/logic game, individuals with no biological or even scientific background are able to make meaningful contributions to areas such as fundamental biochemistry, diagnostics, therapeutics, material design, and more. Players are asked to use RNA’s four nucleotides to design a pattern that will form into a specific shape (or shapes) under various conditions. Over time, these design challenges have become increasingly complex, and reflect a wide variety of mechanics in nature. Initially, players design 3 in a simulated environment for studying and playing with RNA designs that use industry-standard software to predict likely RNA foldings. As they gain experience, players reach the virtual lab and a series of lab challenges. Players create molecular designs for specific biomedical and biotechnology targets. At Stanford University, a partnered wet lab manufactures and tests the designs voted on by the community and provides feedback to the players about how well each design performed. Players then as individuals, and a community, analyze results and use this information to create better designs. Notable Achievements Projects ● Fundamental Research in Riboswitch Design - Successfully designed RNAs which change structure based in the presence of external molecules; a necessary precondition for other initiatives to create diagnostics as well as treatment controls and safety mechanisms ● OpenVaccine - A challenge to develop a safe mRNA vaccine optimized for stability and efficacy, as well as future challenges to create new antivirals ● OpenTB - Designing a fast, low-cost, easily-deployable diagnostic to help identify early-stage tuberculosis, one of the world’s largest public health crises ● OpenCRISPR - Improving safety of the CRISPR gene editing technology by allowing it to be turned on and off on demand ● Ribosome Challenge - Stabilizing the ribosome to allow for further work in ribosome engineering, including the ability to develop novel materials that may be costly or impossible to produce by other methods Papers ● RNA design rules from a massive open laboratory - The culmination of Eterna’s initial research, creating a novel RNA design algorithm (Eternabot) from player analyses and strategies ● Principles for Predicting RNA Secondary Structure Design Difficulty - Creation of a benchmark for RNA design algorithms, now widely used. First paper in the scientific literature to include videogame players as lead authors ● Evidence of an Unusual Poly(A) RNA Signature Detected by High-throughput Chemical Mapping - Discovery of a previously unrecognized experimental artifact, first paper in the scientific literature to include only videogame players as authors ● An unexpectedly effective Monte Carlo technique for the RNA inverse folding problem - RNA design algorithm developed by an Eterna player, far surpassing existing algorithms developed by expert groups ● SentRNA and EternaBrain - RNA design algorithms developed from Eterna player solutions and move history 4 Press Coverage ● New York Times ● CNN ● Wired ● NOVA ● Wall Street Journal History Origins The idea for Eterna was inspired by Foldit, a successful citizen science game centered around protein folding. At RosettaCon 2009, Carnegie Mellon University (CMU) graduate student Jeehyung Lee first presented the idea publicly as a modified version of Foldit that could be used for RNA after discussions in preceding months with his advisor Dr. Adrien Treuille (one of the creators of Foldit), and Dr. Rhiju Das (computational biochemist at Stanford University). They decided they wanted to create a new type of citizen science game that incorporated an online open laboratory into the experience. The project started taking shape through a software development team led by Treuille and Lee at CMU and an experimental biochemistry lab team at Stanford led by Das. Eterna went into beta testing at the end of 2010, and fully launched in January 2011. Since then, the project has continued to grow and evolve to tackle bigger and bigger challenges. Science Eterna’s initial experiments were done using SHAPE chemical mapping, verifying whether or not each RNA base was either bound or unbound as desired. Initial puzzles were “single state”, but evolved to also include “switch” puzzles, where the objective is to design an RNA that is able to change shape after the introduction of a small molecule (in early experiments, FMN). The focus of the research was to attempt to deduce principles of how RNA folds and how to create successful designs. Players proved to outperform existing algorithms, and provided their strategies through Eterna’s “Strategy Market'' for the creation of a novel design algorithm, Eternabot. These initial studies were presented in the paper RNA design rules from a massive open laboratory published in PNAS, with Eterna players listed as a consortium author. In 2013, the Das Laboratory developed new synthesis techniques allowing for high-throughput synthesis and analysis of RNA designs - dubbed “Cloud Lab.” Instead of 8, or at most tens, of designs synthesized per lab round, the Das Laboratory increased their capacity to hundreds or thousands of player designs at a time. The “Cloud Lab” expansion also allowed for something novel - the ability for players to propose their own experiments, which was done throughout the lifetime of the Cloud Lab. These efforts would help to lead to the publication of the first paper 5 with Eterna players as lead authors, Principles for Predicting RNA Secondary Structure Design Difficulty, and a paper with only Eterna players as authors, Evidence of an Unusual Poly(A) RNA Signature Detected by High-throughput Chemical Mapping. In 2015, the Das Laboratory introduced a new experimental method: the RNA Array (also referred to “imaging stations”). The new method used the binding of fluorescent molecules (the MS2 viral coat protein) to RNA as a readout to determine the effectiveness of riboswitches, RNA molecules whose structures change when they bind other molecules. With the ability to test thousands of players’ designs for RNA switch puzzles at a time, Eterna players began to tackle some of their hardest challenges yet: building RNA computers. And this time, instead of small molecules, players would be attempting to bind oligonucleotides (oligos), small pieces of RNA or DNA. Eterna players quickly demonstrated their ability to design logic gates and ratiometric calculators using RNA binding to multiple oligos. The testing of multi-state RNAs on the RNA Array was perfect preparation for Eterna’s first medically-relevant challenges, OpenTB, aiming to create a fast, low-cost, easily-deployable diagnostic for tuberculosis,
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