Recent discoveries of biologically fundamental non-coding RNAs are inspiring novel antibacterial, antitumor, and antiviral therapies that might disable or manipulate the molecules involved. However, in silico folding models cannot yet confidently predict RNA conformations in vitro, slowing the development of these potentially life-saving efforts. To develop the next-generation of robust and rigorously tested RNA bioengineering rules, we are exploring an unconventional strategy: last year, we released a citizen science project enabling non-experts to develop and test folding hypotheses in internet-scale RNA design competitions that are rigorously scored through wet-lab feedback. Launched in early 2011, the 25,000-player EteRNA project already outperforms existing previous methods for designing novel RNA structures which fold properly in vitro. The critical next stage is to sustain and formalize the RNA nanoengineering insights preserved within the EteRNA community. We propose steps to consolidate the community's experimentally validated human computation into a suite of automated design algorithms (EteRNAbot) usable by the entire RNA nanoenginering community. Beyond compiling such cases, we will crowd-source the rigorous search and resolution of in silico and in vitro design failures with thousands of new experiments. Finally, we will deploy the EteRNA- 3D interface for both expert and crowd-sourced three-dimensional design, leveraging a growing database of 3D building blocks and the ROSETTA RNA folding/design algorithms. We will evaluate success in each aim via synthesis and single-nucleotide- resolution chemical mapping of novel designs through our high-throughput wet-lab pipeline. More generally, we will evaluate success by assessing the extent of utilization and citation of the automated design tools;the extent of mining and citation of the publically available RNA Mapping Database generated for the project;and the adoption of the EteRNA paradigm for Internet-scale scientific discovery in biomedical computation problems beyond nucleic acid design.

Public Health Relevance

RNA molecules play fundamental roles in transmitting and regulating genetic information in all living systems, including disease-causing bacteria, retroviruses like HIV, and tumor cells. New potentially life-saving therapies that target these RNAs are being hindered by the slow rate of designing RNA sequences with new folds and interactions. The proposed research seeks to resolve this bottleneck by enabling tens of thousands of citizen scientists to hypothesize new rules for robust RNA design and to test these ideas via high-throughput RNA synthesis and chemical experimentation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM100953-03
Application #
8668102
Study Section
Macromolecular Structure and Function D Study Section (MSFD)
Program Officer
Preusch, Peter
Project Start
2012-06-05
Project End
2016-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
3
Fiscal Year
2014
Total Cost
$455,212
Indirect Cost
$84,883
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Lee, Jeehyung; Kladwang, Wipapat; Lee, Minjae et al. (2014) RNA design rules from a massive open laboratory. Proc Natl Acad Sci U S A 111:2122-7
Lipfert, Jan; Skinner, Gary M; Keegstra, Johannes M et al. (2014) Double-stranded RNA under force and torque: similarities to and striking differences from double-stranded DNA. Proc Natl Acad Sci U S A 111:15408-13
Kladwang, Wipapat; Mann, Thomas H; Becka, Alex et al. (2014) Standardization of RNA chemical mapping experiments. Biochemistry 53:3063-5
Seetin, Matthew G; Kladwang, Wipapat; Bida, John P et al. (2014) Massively parallel RNA chemical mapping with a reduced bias MAP-seq protocol. Methods Mol Biol 1086:95-117