Engineering ligand-responsive RNA controllers in yeast through the assembly of RNase III tuning modules

The programming of cellular networks to achieve new biological functions depends on the development of genetic tools that link the presence of a molecular signal to gene-regulatory activity. Recently, a set of engineered RNA controllers was described that enabled predictable tuning of gene expression in the yeast Saccharomyces cerevisiae through directed cleavage of transcripts by an RNase III enzyme, Rnt1p. Here, we describe a strategy for building a new class of RNA sensing-actuation devices based on direct integration of RNA aptamers into a region of the Rnt1p hairpin that modulates Rnt1p cleavage rates. We demonstrate that ligand binding to the integrated aptamer domain is associated with a structural change sufficient to inhibit Rnt1p processing. Three tuning strategies based on the incorporation of different functional modules into the Rnt1p switch platform were demonstrated to optimize switch dynamics and ligand responsiveness. We further demonstrated that these tuning modules can be implemented combinatorially in a predictable manner to further improve the regulatory response properties of the switch. The modularity and tunability of the Rnt1p switch platform will allow for rapid optimization and tailoring of this gene control device, thus providing a useful tool for the design of complex genetic networks in yeast

Science To Support DOE Site Cleanup: The Pacific Northwest National Laboratory Environmental Management Science Program Awards

Pacific Northwest National Laboratory (PNNL) was awarded ten Environmental Management Science Program (EMSP) research grants in fiscal year 1996, six in fiscal year 1997, nine in fiscal year 1998, seven in fiscal year 1999, and five in fiscal year 2000. All of the fiscal year 1996 award projects have published final reports. The 1997 and 1998 award projects have been completed or are nearing completion. Final reports for these awards will be published, so their annual updates will not be included in this document. This section summarizes how each of the 1999 and 2000 grants address significant U.S. Department of Energy (DOE) cleanup issues, including those at the Hanford Site. The technical progress made to date in each of these research projects is addressed in more detail in the individual progress reports contained in this document. The 1999 and 2000 EMSP awards at PNNL are focused primarily in two areas: Tank Waste Remediation, and Soil and Groundwater Cleanup

DNA Aptamers against the Lup an 1 Food Allergen

Using in vitro selection, high affinity DNA aptamers to the food allergen Lup an 1, ß-conglutin, were selected from a pool of DNA, 93 bases in length, containing a randomised sequence of 49 bases. ß-conglutin was purified from lupin flour and chemically crosslinked to carboxylated magnetic beads. Peptide mass fingerprinting was used to confirm the presence of the ß-conglutin. Single stranded DNA was generated from the randomised pool using T7 Gene 6 Exonuclease and was subsequently incubated with the magnetic beads and the captured DNA was released and amplified prior to a further round of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Evolution was monitored using enzyme linked oligonucleotide assay and surface plasmon resonance. Once a plateau in evolution was reached, the isolated DNA sequences were cloned and sequenced. The consensus motif was identified via alignment of the sequences and the affinities of these sequences for immobilised ß-conglutin were determined using surface plasmon resonance. The selected aptamer was demonstrated to be highly specific, showing no cross-reactivity with other flour ingredients or with other conglutin fractions of lupin. The secondary structures of the selected aptamers were predicted using m-fold. Finally, the functionality of the selected aptamers was demonstrated using a competitive assay for the quantitative detection of ß-conglutin. . Future work will focus on structure elucidation and truncation of the selected sequences to generate a smaller aptamer for application to the analysis of the Lup an 1 allergen in foodstuffs

Thermodynamics and Kinetics of Adaptive Binding in the Malachite Green RNA Aptamer

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/bi400549sAdaptive binding, the ability of molecules to fold themselves around the structure of a ligand and thereby incorporating it into their three-dimensional fold, is a key feature of most RNA aptamers. The malachite green aptamer (MGA) has been shown to bind several closely related triphenyl dyes with planar and nonplanar structures in this manner. Competitive binding studies using isothermal titration calorimetry and stopped flow kinetics have been conducted with the aim of understanding the adaptive nature of RNA–ligand interaction. The results of these studies reveal that binding of one ligand can reduce the ability of the aptamer pocket to adapt to another ligand, even if this second ligand has a significantly higher affinity to the free aptamer. A similar effect is observed in the presence of Mg2+ ions which stabilize the binding pocket in a more ligand bound-like conformation.National Science and Engineering Research Council (NSERC) [326911-2009]Canada Foundation for Innovation (CFI

Synthetic biology: Understanding biological design from synthetic circuits

An important aim of synthetic biology is to uncover the design principles of natural biological systems through the rational design of gene and protein circuits. Here, we highlight how the process of engineering biological systems — from synthetic promoters to the control of cell–cell interactions — has contributed to our understanding of how endogenous systems are put together and function. Synthetic biological devices allow us to grasp intuitively the ranges of behaviour generated by simple biological circuits, such as linear cascades and interlocking feedback loops, as well as to exert control over natural processes, such as gene expression and population dynamics