Crowdsourced RNA design discovers diverse, reversible, efficient, self-contained molecular sensors

Abstract

118 words; 125 words max) Internet-based scientific communities promise a means to apply distributed, diverse human intelligence towards previously intractable scientific problems. However, current implementations have not allowed communities to propose experiments to test all emerging hypotheses at scale or to modify hypotheses in response to experiments. We report high-throughput methods for molecular characterization of nucleic acids that enable the large-scale videogame-based crowdsourcing of functional RNA sensor design, followed by high-throughput functional characterization. Iterative design testing of thousands of crowdsourced RNA sensor designs produced near-thermodynamically optimal and reversible RNA switches that act as self-contained molecular sensors and couple five distinct small molecule inputs to three distinct protein binding and fluorogenic outputs-results that surpass computational and expert-based design. This work represents a new paradigm for widely distributed experimental bioscience. One Sentence Summary: (40 characters)Online community discovers standalone RNA sensors. Main Text:A number of recent platforms have demonstrated the engagement, via the internet, of vast untapped human cognitive potential, transforming the scale by which scientific questions can be approached with human intelligence. However, previous work has only allowed "citizen scientists" to process data already collected 1,2 , to interface with purely computational methods to

RNA secondary structure packages evaluated and improved by high-throughput experiments

“…As with the chemical mapping datasets, each riboswitch dataset was filtered to exclude highly similar sequences (Figure S2, Table S6). These riboswitches came from two sources: the first consisted of 4,849 riboswitches (after filtering) designed by citizen scientists on Eterna 33 . The second consisted of 2,509 riboswitches (after filtering) designed fully computationally using the RiboLogic package, 34 S8.…”

Crowdsourcing and open innovation in drug discovery: recent contributions and future directions

“…Not only has the problem of RNA folding been abstracted to an engaging game open to non-subject matter experts, but the translation of the creation of digital insight has also been made manifest in the real world 49 , 50 . Recently, this approach was brought to scale, leveraging crowdsourced RNA design to enable the discovery of reversible, efficient, and diverse self-contained molecular sensors [51] .…”

High-Throughput Selection and Characterisation of Aptamers on Optical Next-Generation Sequencers

“…Prot-MaP, for example, enables the display of proteins with a length of up to 200 amino acids, whereas peptide array platforms are typically limited to about 16 amino acids [32]. Therefore, HiTS-FLIP is suitable to analyse even complex structures, such as ribozymes and riboswitches [56], making HiTS-FLIP a more versatile technology. In addition, depending on the aptamers analysed, HiTS-FLIP can be more cost-effective than microarrays, since the cost of this technology is similar to that of regular Illumina sequencing [32].…”

“…The same chemistry can be exploited for fluorescent labelling in many cases. Furthermore, different riboswitches, combined from two aptamers-one for a non-fluorescent small molecule ('sensor') and one for a fluorogenic molecule ('reporter')-were already analysed using HiTS-FLIP [56]. It would be conceivable to assay aptamers against non-fluorescent small molecules, via such an existing riboswitch system, if the corresponding aptamer part of the riboswitch is mutated.…”

“…Because of the aspects outlined, HiTS-FLIP is particularly easily applicable but not limited to investigating interactions with proteins. For example, HiTS-FLIP experiments have been successfully performed with transcription factors [27,40], RNA-binding proteins [30,[51][52][53], Cas9 and Cas3 [41,42], antibodies [32,33], and other proteins [28,[30][31][32][33]51], as well as with the fluorogenic small molecules DFHBI [33] and malachite green [56], the peptide insulin [28], and RNA [54,55]. Mamet et al even demonstrated that the affinity of particular sequences towards human tumour cells can be determined via HiTS-FLIP, based on their 'bound fraction'-the ratio of the number of times one of the sequences' clusters co-localized with a bound cell to the total number of clusters of that particular sequence [29].…”