Modular, programmable RNA sensing using ADAR editing in living cells.

With the increasing availability of single-cell transcriptomes, RNA signatures offer a promising basis for targeting living cells. Molecular RNA sensors would enable the study of and therapeutic interventions for specific cell types/states in diverse contexts, particularly in human patients and non-model organisms. Here we describe a modular, programmable system for live RNA sensing using adenosine deaminases acting on RNA (RADAR). We validate, and then expand, our basic design, characterize its performance, and analyze its compatibility with human and mouse transcriptomes. We identify strategies to boost output levels and improve the dynamic range. Additionally, we show that RADAR enables compact AND logic. In addition to responding to transcript levels, RADAR can distinguish disease-relevant sequence alterations of transcript identities, such as point mutations and fusions. Finally, we demonstrate that RADAR is a self-contained system with the potential to function in diverse organisms.

Formation of synthetic RNA protein granules using engineered phage-coat-protein -RNA complexes.

Liquid-solid transition, also known as gelation, is a specific form of phase separation in which molecules cross-link to form a highly interconnected compartment with solid - like dynamical properties. Here, we utilize RNA hairpin coat-protein binding sites to form synthetic RNA based gel-like granules via liquid-solid phase transition. We show both in-vitro and in-vivo that hairpin containing synthetic long non-coding RNA (slncRNA) molecules granulate into bright localized puncta. We further demonstrate that upon introduction of the coat-proteins, less-condensed gel-like granules form with the RNA creating an outer shell with the proteins mostly present inside the granule. Moreover, by tracking puncta fluorescence signals over time, we detected addition or shedding events of slncRNA-CP nucleoprotein complexes. Consequently, our granules constitute a genetically encoded storage compartment for protein and RNA with a programmable controlled release profile that is determined by the number of hairpins encoded into the RNA. Our findings have important implications for the potential regulatory role of naturally occurring granules and for the broader biotechnology field.

A Cell-Free Assay for Rapid Screening of Inhibitors of hACE2-Receptor-SARS-CoV-2-Spike Binding.

We present a cell-free assay for rapid screening of candidate inhibitors of protein binding, focusing on inhibition of the interaction between the SARS-CoV-2 Spike receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (hACE2). The assay has two components: fluorescent polystyrene particles covalently coated with RBD, termed virion-particles (v-particles), and fluorescently labeled hACE2 (hACE2F) that binds the v-particles. When incubated with an inhibitor, v-particle-hACE2F binding is diminished, resulting in a reduction in the fluorescent signal of bound hACE2F relative to the noninhibitor control, which can be measured via flow cytometry or fluorescence microscopy. We determine the amount of RBD needed for v-particle preparation, v-particle incubation time with hACE2F, hACE2F detection limit, and specificity of v-particle binding to hACE2F. We measure the dose response of the v-particles to known inhibitors. Finally, utilizing an RNA-binding protein tdPP7 incorporated into hACE2F, we demonstrate that RNA-hACE2F granules trap v-particles effectively, providing a basis for potential RNA-hACE2F therapeutics.

Overcoming the design, build, test bottleneck for synthesis of nonrepetitive protein-RNA cassettes.

We apply an oligo-library and machine learning-approach to characterize the sequence and structural determinants of binding of the phage coat proteins (CPs) of bacteriophages MS2 (MCP), PP7 (PCP), and Qß (QCP) to RNA. Using the oligo library, we generate thousands of candidate binding sites for each CP, and screen for binding using a high-throughput dose-response Sort-seq assay (iSort-seq). We then apply a neural network to expand this space of binding sites, which allowed us to identify the critical structural and sequence features for binding of each CP. To verify our model and experimental findings, we design several non-repetitive binding site cassettes and validate their functionality in mammalian cells. We find that the binding of each CP to RNA is characterized by a unique space of sequence and structural determinants, thus providing a more complete description of CP-RNA interaction as compared with previous low-throughput findings. Finally, based on the binding spaces we demonstrate a computational tool for the successful design and rapid synthesis of functional non-repetitive binding-site cassettes.

An Assay for Quantifying Protein-RNA Binding in Bacteria.

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

Synthetic 5' UTRs Can Either Up- or Downregulate Expression upon RNA-Binding Protein Binding.

The construction of complex gene-regulatory networks requires both inhibitory and upregulatory modules. However, the vast majority of RNA-based regulatory "parts" are inhibitory. Using a synthetic biology approach combined with SHAPE-seq, we explored the regulatory effect of RNA-binding protein (RBP)-RNA interactions in bacterial 5' UTRs. By positioning a library of RNA hairpins upstream of a reporter gene and co-expressing them with the matching RBP, we observed a set of regulatory responses, including translational stimulation, translational repression, and cooperative behavior. Our combined approach revealed three distinct states in vivo: in the absence of RBPs, the RNA molecules can be found in either a molten state that is amenable to translation or a structured phase that inhibits translation. In the presence of RBPs, the RNA molecules are in a semi-structured phase with partial translational capacity. Our work provides new insight into RBP-based regulation and a blueprint for designing complete gene-regulatory circuits at the post-transcriptional level.

Designing Bacterial Chemotactic Receptors Guided by Photonic Femtoliter Well Arrays for Quantifiable, Label-Free Measurement of Bacterial Chemotaxis.

Whole cell bioreporters, such as bacterial cells, can be used for environmental and clinical sensing of specific analytes. However, the current methods implemented to observe such bioreporters in the form of chemotactic responses heavily rely on microscope analysis, fluorescent labels, and hard-to-scale microfluidic devices. Herein, we demonstrate that chemotaxis can be detected within minutes using intrinsic optical measurements of silicon femtoliter well arrays (FMAs). This is done via phase-shift reflectometric interference spectroscopic measurements (PRISM) of the wells, which act as silicon diffraction gratings, enabling label-free, real-time quantification of the number of trapped bacteria cells in the optical readout. By generating unsteady chemical gradients over the wells, we first demonstrate that chemotaxis toward attractants and away from repellents can be easily differentiated based on the signal response of PRISM. The lowest concentration of chemorepellent to elicit an observed bacterial response was 50 mM, whereas the lowest concentration of chemoattractant to elicit a response was 10 mM. Second, we employed PRISM, in combination with a computational approach, to rapidly scan for and identify a novel synthetic histamine chemoreceptor strain. Consequently, we show that by using a combined computational design approach, together with a quantitative, real-time, and label-free detection method, it is possible to manufacture and characterize novel synthetic chemoreceptors in Escherichia coli (E. coli).

An in Vivo Binding Assay for RNA-Binding Proteins Based on Repression of a Reporter Gene.

We study translation repression in bacteria by engineering a regulatory circuit that functions as a binding assay for RNA binding proteins (RBP) in vivo. We do so by inducing expression of a fluorescent protein-RBP chimera, together with encoding its binding site at various positions within the ribosomal initiation region (+11-13 nt from the AUG) of a reporter module. We show that when bound by their cognate RBPs, the phage coat proteins for PP7 (PCP) and Qß (QCP), strong repression is observed for all hairpin positions within the initiation region. Yet, a sharp transition to no-effect is observed when positioned in the elongation region, at a single-nucleotide resolution. Employing in vivo Selective 2'-hydroxyl acylation analyzed by primer extension followed by sequencing (SHAPE-seq) for a representative construct, established that in the translationally active state the mRNA molecule is nonstructured, while in the repressed state a structured signature was detected. We then utilize this regulatory phenomena to quantify the binding affinity of the coat proteins of phages MS2, PP7, GA, and Qß to 14 cognate and noncognate binding sites in vivo. Using our circuit, we demonstrate qualitative differences between in vitro to in vivo binding characteristics for various variants when comparing to past studies. Furthermore, by introducing a simple mutation to the loop region for the Qß-wt site, MCP binding is abolished, creating the first high-affinity QCP site that is completely orthogonal to MCP. Consequently, we demonstrate that our hybrid transcriptional-post-transcriptional circuit can be utilized as a binding assay to quantify RNA-RBP interactions in vivo.

Fitness effects of interspecific competition between two species of desert rodents.

Many factors affect individual fitness, but while some factors, such as resource availability, have received strong experimental support, others including interspecific competition have rarely been quantified. Nevertheless, interspecific competition is commonly mentioned in the context of reproductive success and fitness. In general, when reproduction is likely to fail, reproductive suppression may occur. We studied the golden spiny mouse (Acomys russatus) and the common spiny mouse (A. cahirinus; however, recent molecular analysis in spiny mice from Jordan and Sinai suggests this species is A. dimidiatus (Frynta et al., 2010), as a model for the effect of competition on reproduction in four field enclosures: two populated only by A. russatus individuals, and two populated by individuals of both species. In presence of A. cahirinus, fitness of A. russatus was lower: the number of A. rusatus offspring was significantly lower; more males had regressed testes (indicating reproductive depression); more A. russatus young had damaged tails. However, no clear effect was evident in A. russatus female vaginal smear cytology. We conclude that the presence of A. cahirinus impairs fitness and reproductive success of A. russatus. While various direct and/or indirect mechanisms may be responsible for the effect of competition on reproduction, a plausible mechanism is increased use of torpor induced by the presence of A. cahirinus previously documented in A. russatus.

The effect of fasting and body reserves on cold tolerance in 2 pit-building insect predators.

Pit-building antlions and wormlions are 2 distantly-related insect species, whose larvae construct pits in loose soil to trap small arthropod prey. This convergent evolution of natural histories has led to additional similarities in their natural history and ecology, and thus, these 2 species encounter similar abiotic stress (such as periodic starvation) in their natural habitat. Here, we measured the cold tolerance of the 2 species and examined whether recent feeding or food deprivation, as well as body composition (body mass and lipid content) and condition (quantified as mass-to-size residuals) affect their cold tolerance. In contrast to other insects, in which food deprivation either enhanced or impaired cold tolerance, prolonged fasting had no effect on the cold tolerance of either species, which had similar cold tolerance. The 2 species differed, however, in how cold tolerance related to body mass and lipid content: although body mass was positively correlated with the wormlion cold tolerance, lipid content was a more reliable predictor of cold tolerance in the antlions. Cold tolerance also underwent greater change with ontogeny in wormlions than in antlions. We discuss possible reasons for this lack of effect of food deprivation on both species' cold tolerance, such as their high starvation tolerance (being sit-and-wait predators).

Group dynamics and relocation decisions of a trap-building predator are differentially affected by biotic and abiotic factors.

Most habitats in nature are heterogeneous, incorporating favorable and unfavorable microhabitats for different animals, based on their ecological niche. Unsuitable microhabitats have negative consequences for individual growth and survival. Animals, therefore, should fine-tune their location within the habitat by dispersing away from such microhabitats. We studied the suitability of different constant microhabitat conditions for wormlion larvae, a trap-building predator, tested in groups under laboratory conditions. Wormlions construct pit-traps in loose soil and capture small arthropod prey. As wormlions occur in high densities in nature, testing in groups is thus more indicative of their natural behavior than testing individuals. Wormlions responded strongly to biotic conditions-high conspecific density, starvation, and large body mass of conspecifics-by either increasing pit-relocation events or moving away from the microhabitat center to the periphery of the arena, probably opting for a way out. In other instances, individuals increased their distance to the nearest neighbor, thereby changing the spatial pattern toward a more regular pattern, potentially indicating interference competition. The only abiotic condition apparently perceived by wormlions as unsuitable was shallow sand, which led to frequent relocations. The two other abiotic factors-illumination and sand particle size-had no observable effect on behavior, although wormlions in nature always occur under shade in fine sand, and prefer both shade and fine sand particle size under laboratory conditions when given a choice. Under the fine spatial scale of the present experiment, biotic factors appear to be more influential than abiotic ones.