Compact RNA sensors for increasingly complex functions of multiple inputs.

Designing single molecules that compute general functions of input molecular partners represents a major unsolved challenge in molecular design. Here, we demonstrate that high-throughput, iterative experimental testing of diverse RNA designs crowdsourced from Eterna yields sensors of increasingly complex functions of input oligonucleotide concentrations. After designing single-input RNA sensors with activation ratios beyond our detection limits, we created logic gates, including challenging XOR and XNOR gates, and sensors that respond to the ratio of two inputs. Finally, we describe the OpenTB challenge, which elicited 85-nucleotide sensors that compute a score for diagnosing active tuberculosis, based on the ratio of products of three gene segments. Building on OpenTB design strategies, we created an algorithm Nucleologic that produces similarly compact sensors for the three-gene score based on RNA and DNA. These results open new avenues for diverse applications of compact, single molecule sensors previously limited by design complexity.

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

Internet-based scientific communities promise a means to apply distributed, diverse human intelligence toward 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 video game­based crowdsourcing of RNA sensor design, followed by high-throughput functional characterization. Iterative design testing of thousands of crowdsourced RNA sensor designs produced near­thermodynamically 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. This work suggests a paradigm for widely distributed experimental bioscience.

Computational design of three-dimensional RNA structure and function.

RNA nanotechnology seeks to create nanoscale machines by repurposing natural RNA modules. The field is slowed by the current need for human intuition during three-dimensional structural design. Here, we demonstrate that three distinct problems in RNA nanotechnology can be reduced to a pathfinding problem and automatically solved through an algorithm called RNAMake. First, RNAMake discovers highly stable single-chain solutions to the classic problem of aligning a tetraloop and its sequence-distal receptor, with experimental validation from chemical mapping, gel electrophoresis, solution X-ray scattering and crystallography with 2.55 Å resolution. Second, RNAMake automatically generates structured tethers that integrate 16S and 23S ribosomal RNAs into single-chain ribosomal RNAs that remain uncleaved by ribonucleases and assemble onto messenger RNA. Third, RNAMake enables the automated stabilization of small-molecule binding RNAs, with designed tertiary contacts that improve the binding affinity of the ATP aptamer and improve the fluorescence and stability of the Spinach RNA in cell extracts and in living Escherichia coli cells.

Evaluating riboswitch optimality.

Riboswitches are RNA elements that recognize diverse chemical and biomolecular inputs, and transduce this recognition process to genetic, fluorescent, and other engineered outputs using RNA conformational changes. These systems are pervasive in cellular biology and are a promising biotechnology with applications in genetic regulation and biosensing. Here, we derive a simple expression bounding the activation ratio-the proportion of RNA in the active vs. inactive states-for both ON and OFF riboswitches that operate near thermodynamic equilibrium: 1+[I]/KdI, where [I] is the input ligand concentration and KdI is the intrinsic dissociation constant of the aptamer module toward the input ligand. A survey of published studies of natural and synthetic riboswitches confirms that the vast majority of empirically measured activation ratios have remained well below this thermodynamic limit. A few natural and synthetic riboswitches achieve activation ratios close to the limit, and these molecules highlight important principles for achieving high riboswitch performance. For several applications, including "light-up" fluorescent sensors and chemically-controlled CRISPR/Cas complexes, the thermodynamic limit has not yet been achieved, suggesting that current tools are operating at suboptimal efficiencies. Future riboswitch studies will benefit from comparing observed activation ratios to this simple expression for the optimal activation ratio. We present experimental and computational suggestions for how to make these quantitative comparisons and suggest new molecular mechanisms that may allow non-equilibrium riboswitches to surpass the derived limit.

Direct Selection of Fluorescence-Enhancing RNA Aptamers.

RNA aptamers that generate a strong fluorescence signal upon binding a nonfluorescent small-molecule dye offer a powerful means for the selective imaging of individual RNA species. Unfortunately, conventional in vitro discovery methods are not efficient at generating such fluorescence-enhancing aptamers, because they primarily exert selective pressure based on target affinity-a characteristic that correlates poorly with fluorescence enhancement. Thus, only a handful of fluorescence-enhancing aptamers have been reported to date. In this work, we describe a method for converting DNA libraries into "gene-linked RNA aptamer particles" (GRAPs) that each display ∼105 copies of a single RNA sequence alongside the DNA that encodes it. We then screen large libraries of GRAPs in a high-throughput manner using the FACS instrument based directly on their fluorescence-enhancing properties. Using this strategy, we demonstrate the capability to generate fluorescence-enhancing aptamers that produce a variety of different emission wavelengths upon binding the dye of interest.

Advancements in Aptamer Discovery Technologies.

Affinity reagents that specifically bind to their target molecules are invaluable tools in nearly every field of modern biomedicine. Nucleic acid-based aptamers offer many advantages in this domain, because they are chemically synthesized, stable, and economical. Despite these compelling features, aptamers are currently not widely used in comparison to antibodies. This is primarily because conventional aptamer-discovery techniques such as SELEX are time-consuming and labor-intensive and often fail to produce aptamers with comparable binding performance to antibodies. This Account describes a body of work from our laboratory in developing advanced methods for consistently producing high-performance aptamers with higher efficiency, fewer resources, and, most importantly, a greater probability of success. We describe our efforts in systematically transforming each major step of the aptamer discovery process: selection, analysis, and characterization. To improve selection, we have developed microfluidic devices (M-SELEX) that enable discovery of high-affinity aptamers after a minimal number of selection rounds by precisely controlling the target concentration and washing stringency. In terms of improving aptamer pool analysis, our group was the first to use high-throughput sequencing (HTS) for the discovery of new aptamers. We showed that tracking the enrichment trajectory of individual aptamer sequences enables the identification of high-performing aptamers without requiring full convergence of the selected aptamer pool. HTS is now widely used for aptamer discovery, and open-source software has become available to facilitate analysis. To improve binding characterization, we used HTS data to design custom aptamer arrays to measure the affinity and specificity of up to ∼10(4) DNA aptamers in parallel as a means to rapidly discover high-quality aptamers. Most recently, our efforts have culminated in the invention of the "particle display" (PD) screening system, which transforms solution-phase aptamers into "aptamer particles" that can be individually screened at high-throughput via fluorescence-activated cell sorting. Using PD, we have shown the feasibility of rapidly generating aptamers with exceptional affinities, even for proteins that have previously proven intractable to aptamer discovery. We are confident that these advanced aptamer-discovery methods will accelerate the discovery of aptamer reagents with excellent affinities and specificities, perhaps even exceeding those of the best monoclonal antibodies. Since aptamers are reproducible, renewable, stable, and can be distributed as sequence information, we anticipate that these affinity reagents will become even more valuable tools for both research and clinical applications.