-
[show abstract]
[hide abstract]
ABSTRACT: Bacterial regulators of transcriptional elongation are versatile units for building custom genetic switches, as they control the expression of both coding and noncoding RNAs, act on multigene operons and can be predictably tethered into higher-order regulatory functions (a property called composability). Yet the less versatile bacterial regulators of translational initiation are substantially easier to engineer. To bypass this tradeoff, we have developed an adaptor that converts regulators of translational initiation into regulators of transcriptional elongation in Escherichia coli. We applied this adaptor to the construction of several transcriptional attenuators and activators, including a small molecule-triggered attenuator and a group of five mutually orthogonal riboregulators that we assembled into NOR gates of two, three or four RNA inputs. Continued application of our adaptor should produce large collections of transcriptional regulators whose inherent composability can facilitate the predictable engineering of complex synthetic circuits.
Nature Methods 09/2012; · 19.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Our ability to routinely engineer genetic networks for applications is limited by the scarcity of highly specific and non-cross-reacting (orthogonal) gene regulators with predictable behavior. Though antisense RNAs are attractive contenders for this purpose, quantitative understanding of their specificity and sequence-function relationship sufficient for their design has been limited. Here, we use rationally designed variants of the RNA-IN-RNA-OUT antisense RNA-mediated translation system from the insertion sequence IS10 to quantify >500 RNA-RNA interactions in Escherichia coli and integrate the data set with sequence-activity modeling to identify the thermodynamic stability of the duplex and the seed region as the key determinants of specificity. Applying this model, we predict the performance of an additional ~2,600 antisense-regulator pairs, forecast the possibility of large families of orthogonal mutants, and forward engineer and experimentally validate two RNA pairs orthogonal to an existing group of five from the training data set. We discuss the potential use of these regulators in next-generation synthetic biology applications.
Nature Chemical Biology 03/2012; 8(5):447-54. · 14.69 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Non-coding RNAs (ncRNAs) are versatile regulators in cellular networks. While most trans-acting ncRNAs possess well-defined mechanisms that can regulate transcription or translation, they generally lack the ability to directly sense cellular signals. In this work, we describe a set of design principles for fusing ncRNAs to RNA aptamers to engineer allosteric RNA fusion molecules that modulate the activity of ncRNAs in a ligand-inducible way in Escherichia coli. We apply these principles to ncRNA regulators that can regulate translation (IS10 ncRNA) and transcription (pT181 ncRNA), and demonstrate that our design strategy exhibits high modularity between the aptamer ligand-sensing motif and the ncRNA target-recognition motif, which allows us to reconfigure these two motifs to engineer orthogonally acting fusion molecules that respond to different ligands and regulate different targets in the same cell. Finally, we show that the same ncRNA fused with different sensing domains results in a sensory-level NOR gate that integrates multiple input signals to perform genetic logic. These ligand-sensing ncRNA regulators provide useful tools to modulate the activity of structurally related families of ncRNAs, and building upon the growing body of RNA synthetic biology, our ability to design aptamer-ncRNA fusion molecules offers new ways to engineer ligand-sensing regulatory circuits.
Nucleic Acids Research 03/2012; 40(12):5775-86. · 8.03 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Despite great interest in solving RNA secondary structures due to their
impact on function, it remains an open problem to determine structure from
sequence. Among experimental approaches, a promising candidate is the "chemical
modification strategy", which involves application of chemicals to RNA that are
sensitive to structure and that result in modifications that can be assayed via
sequencing technologies. One approach that can reveal paired nucleotides via
chemical modification followed by sequencing is SHAPE, and it has been used in
conjunction with capillary electrophoresis (SHAPE-CE) and high-throughput
sequencing (SHAPE-Seq). The solution of mathematical inverse problems is needed
to relate the sequence data to the modified sites, and a number of approaches
have been previously suggested for SHAPE-CE, and separately for SHAPE-Seq
analysis. Here we introduce a new model for inference of chemical modification
experiments, whose formulation results in closed-form maximum likelihood
estimates that can be easily applied to data. The model can be specialized to
both SHAPE-CE and SHAPE-Seq, and therefore allows for a direct comparison of
the two technologies. We then show that the extra information obtained with
SHAPE-Seq but not with SHAPE-CE is valuable with respect to ML estimation.
06/2011;
-
[show abstract]
[hide abstract]
ABSTRACT: Sequence census methods reduce molecular measurements such as transcript abundance and protein-nucleic acid interactions to counting problems via DNA sequencing. We focus on a novel assay utilizing this approach, called selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), that can be used to characterize RNA secondary and tertiary structure. We describe a fully automated data analysis pipeline for SHAPE-Seq analysis that includes read processing, mapping, and structural inference based on a model of the experiment. Our methods rely on the solution of a series of convex optimization problems for which we develop efficient and effective numerical algorithms. Our results can be easily extended to other chemical probes of RNA structure, and also generalized to modeling polymerase drop-off in other sequence census-based experiments.
Proceedings of the National Academy of Sciences 06/2011; 108(27):11069-74. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems.
Proceedings of the National Academy of Sciences 06/2011; 108(27):11063-8. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Geophagy has been hypothesized to be an adaptive behavior, either as a means to allay nutrient deficiency or to protect against ingested pathogens and toxins. Others have proposed that geophagy is non-adaptive, occurring either to allay hunger or as an epiphenomenon of nutrient deficiencies. This paper evaluates these hypotheses using 482 published cultural-level accounts of human geophagy and 330 accounts of geophagy among 297 species of mammals, birds, and reptiles. Information was extracted from reports of human geophagy to permit statistical analysis; reports of non-human geophagy were tabulated. Human geophagy did not parallel changes in nutrient requirements, occurred most frequently among children and pregnant women and in tropical areas (where pathogen densities are highest), and was associated with ingestion of toxic substances and gastrointestinal distress. Earth ingested by humans was craved and carefully selected and prepared; it had high clay content, but few bioavailable mineral nutrients. In primates, geophagy was associated with both protection from toxins and obtaining nutrients, whereas in other vertebrates it was associated mainly with obtaining nutrients. Our results indicate that human geophagy is best explained as providing protection from dietary chemicals, parasites, and pathogens, whereas animal geophagy may involve both micronutrient acquisition and protection.
The Quarterly Review of Biology 06/2011; 86(2):97-120. · 7.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules.
Proceedings of the National Academy of Sciences 05/2011; 108(21):8617-22. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Our current ability to engineer biological circuits is hindered by design cycles that are costly in terms of time and money, with constructs failing to operate as desired, or evolving away from the desired function once deployed. Synthetic biologists seek to understand biological design principles and use them to create technologies that increase the efficiency of the genetic engineering design cycle. Central to the approach is the creation of biological parts--encapsulated functions that can be composited together to create new pathways with predictable behaviors. We define five desirable characteristics of biological parts--independence, reliability, tunability, orthogonality and composability, and review studies of small natural and synthetic biological circuits that provide insights into each of these characteristics. We propose that the creation of appropriate sets of families of parts with these properties is a prerequisite for efficient, predictable engineering of new function in cells and will enable a large increase in the sophistication of genetic engineering applications.
Current opinion in microbiology 12/2008; 11(6):567-73. · 7.87 Impact Factor
