Metabolic engineering of plants using a disarmed potyvirus vector

Tesis por compendio[EN] Plant viruses are obligate intracellular parasites which were used to develop recombinant plant virus vectors to express heterologous proteins and to modify endogenous metabolic pathways of natural products in plants. The main limitation of many plant virus-based systems is the difficulty to co-express various heterologous proteins in the same cell with proper subcellular localization, which is a crucial question in metabolic engineering. This work provides a solution to overcome this problem by using a potyvirus-based vector system. Potyviruses (genus Potyvirus, family Potyviridae) are plus-strand single-stranded RNA viruses, which have a genome expression strategy that allows the equimolar production of most viral proteins. On the basis of an infectious clone of Tobacco etch virus (TEV), Bedoya et al. (2010) developed an expression system in which the RNA-dependent RNA polymerase (NIb) gene was replaced by an expression cassette, harboring several heterologous proteins. This viral vector was able to express three fluorescent proteins with nucleocytoplasmic localization in equimolar amounts in transgenic tobacco plants in which NIb was supplemented in trans. Despite of the apparent simplicity of potyvirus genome expression strategy, foreign cDNA insertion is a complicated task. Thus, our first goal was to analyze the effect of gene insertion on TEV genome stability. As a result of this work, a novel insertion position was discovered at the amino-terminal end of the potyvirus polyprotein, which opened the possibility to explore new questions of recombinant protein expression. Since metabolic pathways are highly compartmentalized, proper subcellular targeting of enzymes is an essential task. Thus, our second objective centralized on the subcellular targeting of expressed proteins from the TEV-based viral vector. cDNAs coding for the green fluorescent protein (GFP) fused to chloroplast, nucleus and mitochondria targeting signal sequences were inserted into the newly described amino-terminal insertion position or into an internal site, replacing the NIb cistron. Our results showed that for protein delivery to chloroplasts and mitochondria, foreign genes have to be inserted at the amino-terminal site of the viral vector, but for nuclear delivery, both insertion positions are suitable. The last objective of this work was to investigate whether the potyvirus-based vector was able to express an entire heterologous multistep biosynthetic pathway in plant cells. For this aim we purposed to produce lycopene, a plant pigment with health promoting properties. To do so, we inserted cDNAs coding for the enzymes of a three-step metabolic pathway of bacterial origin into the potyvirus-based vector. Infected tobacco plants developed orange symptoms indicating lycopene accumulation, which was confirmed by high-performance liquid chromatography analysis and microscopy observations. Our results also illustrated that the sole expression of Pantoea ananatis phytoene synthase, crtB, is enough to induce carotenoid accumulation, conferring yellow coloration to the infected tissue and serves as reporter system to visually track viral infection in several plant species.[ES] Los virus de plantas son parásitos intracelulares obligados que han sido utilizados para desarrollar vectores virales y expresar proteínas heterólogas y modificar rutas metabólicas endógenas de productos naturales. La principal limitación de muchos sistemas basados en virus de plantas es la dificultad de coexpresar diversas proteínas heterólogas en la misma célula con la localización subcelular apropiada, lo cual es una cuestión crucial en ingeniería metabólica. Este trabajo presenta una solución para superar este problema mediante el uso de un vector viral basado en un potyvirus. Los potyvirus (género Potyvirus, familia Potyviridae) son virus de RNA de cadena positiva simple que tienen una estrategia de expresión génica que permite la producción de la mayoría de las proteínas virales en cantidades equimolares. Basado en un clon infeccioso del virus del grabado del tabaco (Tobacco etch virus, TEV) Bedoya et al. (2010) desarrollaron un sistema de expresión en el que el gen de la RNA polimerasa dependiente de RNA (NIb) fue sustituido por un casete de expresión, que albergaba varias proteínas heterólogas. Este vector viral fue capaz de expresar tres proteínas fluorescentes con localización nucleocitoplásmica en cantidades equimolares en plantas de tabaco transgénicas que complementaban el cistron NIb en trans. A pesar de la aparente simplicidad de la estrategia de expresión génica de los potyvirus, la inserción de un cDNA foráneo es una tarea complicada. Por lo tanto, nuestro primer objetivo fue analizar el efecto de la inserción en la estabilidad del genoma de TEV. Como resultado de este trabajo, descubrimos una nueva posición de inserción en el extremo amino-terminal de la poliproteína viral que nos permitió explorar otras cuestiones sobre la expresión de proteínas recombinantes. Dado que las vías metabólicas son muy compartimentalizadas, la adecuada localización subcelular de enzimas es una tarea esencial en ingeniería metabólica. Por eso, nuestro segundo objetivo se centró en la distribución de las proteínas heterológas expresadas con el vector viral a diferentes orgánulos subcelulares. cDNAs que codificaban la proteína fluorescente verde (green fluorescent protein, GFP) fusionada a péptidos señal se insertaron en la nueva posición amino-terminal y en un sitio interno, sustituyendo el cistrón NIb, para enviarla al cloroplasto, núcleo y a la mitocondria. Nuestros resultados mostraron que para la distribución de proteínas al cloroplasto y mitocondria, los genes foráneos deben ser insertados en el sitio amino-terminal del vector viral, pero para la distribución nuclear, ambas posiciones son adecuadas. El último objetivo de este trabajo fue estudiar si el vector viral basado en potyvirus es capaz de expresar una ruta biosíntética de múltiples pasos en células vegetales. Para ello nos propusimos producir licopeno, un pigmento vegetal con propiedades beneficiosas para la salud humana. Para ello, insertamos un cDNA que codificaba las enzimas de una ruta metabólica de tres pasos de origen bacteriano en el vector viral. Las plantas de tabaco infectadas con el vector viral desarrollaron síntomas de color naranja indicando la acumulación de licopeno, que fue confirmado por análisis de cromatografía líquida de alta eficacia y observaciones de microscopía. Nuestros resultados también ilustraron que la sola expresión de la fitoeno sintasa de Pantonea ananatis, crtB, es suficiente para inducir la acumulación de carotenoides que confieren una coloración amarilla al tejido infectado y sirve como sistema reportero visual en varias especies de plantas.[CA] Els virus de plantes són paràsits intracel·lulars obligats que han estat utilitzats per a desenvolupar vectors virals i expressar proteïnes heteròlogues y modificar rutes metabòliques endògenes de productes naturals silenciant certs gens o expressant factors de transcripció i enzims metabòlics. La principal limitació de molts sistemes basats en virus de plantes és la dificultat de coexpressar diverses proteïnes heteròlogues en la mateixa cèl·lula amb la localització subcel·lular apropiada, cosa que és una qüestió crucial en enginyeria metabòlica. Aquest treball presenta una solució per a superar aquest problema mitjançant l'ús d'un vector viral basat en un potyvirus. Els potyvirus (gènere Potyvirus, família Potyviridae) són virus d'RNA de cadena positiva simple que tenen una estratègia d'expressió gènica que permet la producció de la majoria de les proteïnes virals en quantitats equimolars. Basat en un clon infecciós del virus del gravat del tabac (Tobacco etch virus, TEV) Bedoya et al. (2010) van desenvolupar un sistema d'expressió en el qual el gen de l'RNA polimerasa depenent d'RNA (NIb) va ser substituït per un casset d'expressió, que albergava diverses proteïnes heteròlogues. Aquest vector viral va ser capaç d'expressar tres proteïnes fluorescents amb localització nucleocitoplàsmica en quantitats equimolars en plantes de tabac transgèniques que complementaven el cistró NIb en trans. Malgrat l'aparent simplicitat de l'estratègia d'expressió gènica dels potyvirus, la inserció d'un cDNA forà és una tasca complicada. Per tant, el nostre primer objectiu va ser analitzar l'efecte de la inserció en l'estabilitat del genoma de TEV. Com a resultat d'aquest treball, hem descobert una nova posició d'inserció en l'extrem amino terminal de la poliproteïna viral que ens va permetre explorar altres qüestions sobre l'expressió de proteïnes recombinants. Atès que les vies metabòliques són molt compartimentalitzades, l'adequada localització subcel·lular d'enzims és una tasca essencial en enginyeria metabòlica. Per açò, el nostre segon objectiu es va centrar en la distribució de les proteïnes heteròlogues expressades amb el vector viral a diferents orgànuls subcelul·lars. cDNAs que codificaven la proteïna fluorescent verda (green fluorescent protein, GFP) fusionada a pèptids senyal es van inserir en la nova posició amino terminal i en un lloc intern, substituint el cistró NIb, per a enviar-la al cloroplast, nucli i al mitocondri. Els nostres resultats van mostrar que per a la distribució de proteïnes al cloroplast i mitocondri, els gens forans han de ser inserits en el lloc amino terminal del vector viral, però per a la distribució nuclear, ambdues posicions són adequades. El lloc amino terminal va resultar ser més adequat per a produir quantitats més grans de proteïnes recombinants, però el lloc d'inserció intern va demostrar ser més estable. Sobre la base d'aquests resultats, hem sigut capaços de distribuir dues proteïnes fluorescents diferents als cloroplasts i nuclis des d'un únic vector viral. L'últim objectiu d'aquest treball va ser estudiar si el vector viral basat en potyvirus és capaç d'expressar una ruta biosintètica de múltiples passos en cèl·lules vegetals. Per açò ens vam proposar produir licopè, un pigment vegetal amb propietats beneficioses per a la salut humana. Per això inserírem un cDNA que codificaba els tres enzims de una ruta metabòlica de tres passos d'origen bacterià en el vector viral. Les plantes de tabac infectades amb el vector viral van desenvolupar símptomes de color taronja indicant l'acumulació de licopè, que va ser confirmat per anàlisi de cromatografia líquida d'alta eficàcia i observacions de microscòpia. Els nostres resultats també van il·lustrar que la sola expressió de fitoè sintasa de Pantonea ananatis, crtB, és suficient per a induir l'acumulació de carotenoides que confereixen una coloraMajer, E. (2016). Metabolic engineering of plants using a disarmed potyvirus vector [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68477TESISCompendi

The role of N- or C-terminal biotinylation in autoantibody recognition of citrullin containing filaggrin epitope peptides in Rheumatoid arthritis

Here, we report on the synthesis, conformational analysis and autoantibody binding properties of new sets of Rheumatoid arthritis (RA) specific biotin-peptide conjugates derived from filaggrin epitope peptides. The biotin with or without a linker was attached to the Cit or Arg containing epitope core (311TXGRS315) or epitope region (306SHQESTXGXSXGRSGRSGS324) peptide (where X = Cit), through an amide bond at the N- or C-terminal of the epitopes. Antibody binding was detected by indirect enzyme-linked immunosorbent assay (ELISA) using sera from RA, Systemic lupus erythematosus (SLE) patients as well as healthy individuals and the secondary structure of conjugates was investigated by electronic circular dichroism (ECD). We found that autoantibodies from RA patients recognize specifically both filaggrin epitope region (306SHQESTXGXSXGRSGRSGS324) and short epitope core (311TXGRS315) peptides. Our data also indicate that the positioning of the biotin label within a peptide sequence can markedly influence the antibody binding, but the length of the linker incorporated has essentially no effect on the recognition. ECD experiments demonstrate that the Arg/Cit change does not influence the solution conformation of the peptide conjugates. However, the presence and position of the biotin moiety has a pronounced effect on the conformation of the 5-mer epitope core peptides, while it doesn’t alter the secondary structure of the 19-mer epitope region peptides

Model-based design of RNA hybridization networks implemented in living cells

[EN] Synthetic gene circuits allow the behavior of living cells to be reprogrammed, and non-coding small RNAs (sRNAs) are increasingly being used as programmable regulators of gene expression. However, sRNAs (natural or synthetic) are generally used to regulate single target genes, while complex dynamic behaviors would require networks of sRNAs regulating each other. Here, we report a strategy for implementing such networks that exploits hybridization reactions carried out exclusively by multifaceted sRNAs that are both targets of and triggers for other sRNAs. These networks are ultimately coupled to the control of gene expression. We relied on a thermo-dynamic model of the different stable conformational states underlying this system at the nucleotide level. To test our model, we designed five different RNA hybridization networks with a linear architecture, and we implemented them in Escherichia coli. We validated the network architecture at the molecular level by native polyacrylamide gel electrophoresis, as well as the network function at the bacterial population and single-cell levels with a fluorescent reporter. Our results suggest that it is possible to engineer complex cellular programs based on RNA from first principles. Because these networks are mainly based on physical interactions, our designs could be expanded to other organisms as portable regulatory resources or to implement biological computations.The Consejo Superior de Investigaciones Cientificas (CSIC) Intramural [grant number 201440I017]; the Ministerio de Economia, Industria y Competitividad (MINECO)/FEDER [grant number BFU2015-66894-P]; and the AXA Research Fund Postdoctoral fellowship to G.R. The predoctoral fellowship [grant number AP2012-3751, MECD] to E.M. The Ministerio de Economia, Industria y Competitividad (MINECO) [grant numbers BIO2014-54269-R, AGL2013-49919-EXP] to J.A.D. The 7th Framework Programme [grant numbers 610730 (EVO-PROG), 613745 (PROMYS)]; the Horizon 2020 Marie Sklodowska-Curie [grant number 642738 (MetaRNA)]; the Engineering and Physical Sciences Research Council (EPSRC) and the Biotechnology and Biological Sciences Research Council (BBSRC) [grant number BB/M017982/1 (WISB centre)]; and the School of Life Sciences (U. Warwick) [startup allocation] to A.J. Funding for open access charge: EPSRC/BBSRC [BB/M017982/1 to A.J.].Rodrigo, G.; Prakash, S.; Shen, S.; Majer, E.; Daros Arnau, JA.; Jaramillo, A. (2017). Model-based design of RNA hybridization networks implemented in living cells. Nucleic Acids Research. 45(16):9797-9808. https://doi.org/10.1093/nar/gkx698S979798084516Ausländer, S., Ausländer, D., Müller, M., Wieland, M., & Fussenegger, M. (2012). Programmable single-cell mammalian biocomputers. Nature, 487(7405), 123-127. doi:10.1038/nature11149Friedland, A. E., Lu, T. K., Wang, X., Shi, D., Church, G., & Collins, J. J. (2009). 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Development of Cyclic NGR Peptides with Thioether Linkage:Structure and Dynamics Determining Deamidation and Bioactivity

NGR peptides that recognize CD13 receptors in tumor neovasculature are of high interest, in particular due to their potential applications in drug targeting. Here we report the synthesis and structural analysis of novel thioether bond-linked cyclic NGR peptides. Our results show that their chemostability (resistance against spontaneous decomposition forming isoAsp and Asp derivatives) strongly depends on both sample handling conditions and structural properties. A significant correlation was found between chemostability and structural measures, such as NHGly−COAsn‑sc distances. The sidechain orientation of Asn is a key determining factor; if it is turned away from HNGly, the chemostability increases. Structure stabilizing factors (e.g., H-bonds) lower their internal dynamics, and thus biomolecules become even more resistant against spontaneous decomposition. The effect of cyclic NGR peptides on cell adhesion was examined in A2058 melanoma cell lines. It was found that some of the investigated peptides gradually increased cell adhesion with long-term characteristics, indicating time-dependent formation of integrin binding isoAsp derivatives that are responsible for the adhesion-inducing effect

Stability and fitness impact of the visually discernible Rosea1 marker in the Tobacco etch virus genome

[EN] Antirrhinum majus Rosea1 (Ros1) is an MYB-related transcription factor that induces anthocyanin biosynthesis in plant tissues, and has been shown to be suitable for visual tracking of virus infection in plants. However, activation of anthocyanin biosynthesis has far reaching effects on plant physiology and could consequently have negative effects on viral replication. Therefore, viruses carrying the Ros1 marker might have a low fitness and consequently rapidly lose the marker. To compare the stability of the Ros1 marker, we generated Tobacco etch virus (TEV) based constructs containing either Ros1 or the enhanced green fluorescent protein (eGFP) between the NIb and CP cistrons (TEV-Ros1 and TEV-eGFP, respectively). We measured the within-host competitive fitness of both viruses by direct competitions with a common competitor during infection of Nicotiana tabacum. The fitness of TEV-Ros1 was significantly lower than that of TEV-eGFP, and both recombinant viruses had a significantly lower fitness than the wild-type virus. Nevertheless, after seven weeks of infection in N. tabacum, similar levels of marker gene instability where found for both viruses. Despite lower fitness of the marked virus, Ros1 is therefore a viable alternative marker for tracking viral infection in plants.The authors thank Santiago F. Elena for useful discussion, Francisca de la Iglesia, Paula Agudo and Veronica Aragones for technical support, and two anonymous reviewers for constructive comments. This research was supported by grants BIO2011-26741 from the Spanish Ministerio de Economia y Competitividad (MINECO) and PROMETEO/2010/019 from Generalitat Valenciana. EM was supported by a predoctoral fellowship (AP2012-3751) from the Spanish Ministerio de Educacion, Cultura y Deporte. MPZ was supported by a 'Juan de la Cierva' postdoctoral contract (JCI-2011-10379) from MINECO.Majer, E.; Daros Arnau, JA.; Zwart, MP. (2013). Stability and fitness impact of the visually discernible Rosea1 marker in the Tobacco etch virus genome. Viruses. 5(9):2153-2168. https://doi.org/10.3390/v5092153S215321685

Photochemical and Structural Studies on Cyclic Peptide Models

L

Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development

[EN] Plastids, the defining organelles of plant cells, undergo physiological and morphological changes to fulfill distinct biological functions. In particular, the differentiation of chloroplasts into chromoplasts results in an enhanced storage capacity for carotenoids with industrial and nutritional value such as beta-carotene (provitamin A). Here, we show that synthetically inducing a burst in the production of phytoene, the first committed intermediate of the carotenoid pathway, elicits an artificial chloroplast-to-chromoplast differentiation in leaves. Phytoene overproduction initially interferes with photosynthesis, acting as a metabolic threshold switch mechanism that weakens chloroplast identity. In a second stage, phytoene conversion into downstream carotenoids is required for the differentiation of chromoplasts, a process that involves a concurrent reprogramming of nuclear gene expression and plastid morphology for improved carotenoid storage. We hence demonstrate that loss of photosynthetic competence and enhanced production of carotenoids are not just consequences but requirements for chloroplasts to differentiate into chromoplasts.We greatly thank Jaume F. Martinez-Garcia and Ralf Welsch for fruitful discussions on the manuscript; Ralf Welsch and Li Li for providing seeds of the Arabidopsis ccd1 ccd4 and ator atorl mutants, respectively; Juan Jose Lopez-Moya and Maria Luisa Domingo-Calap for the gift of the HcProWMV-pGWB702 vector; and M. Rosa Rodriguez-Goberna for excellent technical support. The help of Marti Bernardo, Fidel Lozano, Lidia Jimenez, and members of the CRAG core facilities is also appreciated. This work was funded by the European Regional Development Fund and Spanish Agencia Estatal de Investigacion Grants BIO2017-84041-P, BIO2017-83184-R, BIO2017-90877-REDT, BES-2017-080652, and AGL2017-85563-C2-1-R; Ministry of Education, Culture and Sports Grants AP2012-3751 and FPU16/04054; and Generalitat de Catalunya Grant 2017SGR-710. We also thank the financial support of the European Union's Horizon 2020 (EU-H2020) COST Action CA15136 (EuroCaroten) and Marie S. Curie Action (MSCA) 753301 (Arcatom), the Severo Ochoa Programme for Centres of Excellence in R&D 2016-2019 Grant SEV-2015-0533 and the Generalitat de Catalunya CERCA Programme (to CRAG). B.L. is supported by grants from the CSIRO Synthetic Biology Future Science Platform and Macquarie University. L.M. is supported by La Caixa Foundation PhD INPhINIT Fellowship LCF/BQ/IN18/11660004, which received funding from the EU-H2020 through MSCA Grant 713673. A.R.F. is supported by Deutsche Forschungsgemeinschaft Grant DFG TRR 175.Llorente, B.; Torres-Montilla, S.; Morelli, L.; Florez-Sarasa, I.; Matus, JT.; Ezquerro, M.; D'andrea, L.... (2020). Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development. Proceedings of the National Academy of Sciences of the United States of America (Online). 117(35):21796-21803. https://doi.org/10.1073/pnas.2004405117S21796218031173

Exploring the Dynamics and Mutational Landscape of Riboregulation with a Minimal Synthetic Circuit in Living Cells

[EN] The regulation of gene expression, triggered by conformational changes in RNA molecules, is widely observed in cellular systems. Here, we examine this mode of control by means of a model-based design and construction of a fully synthetic riboregulatory device. We present a theoretical framework that rests on a simple energy model to predict the dynamic response of such a system. Following an equilibrium description, our framework integrates thermodynamic properties-anticipated with an RNA physicochemical model-with a detailed description of the intermolecular interaction. The theoretical calculations are confirmed with an experimental characterization of the action of the riboregulatory device within living cells. This illustrates, more broadly, the predictability of genetic robustness on synthetic systems, and the faculty to engineer gene expression programs from a minimal set of first principles.This work was supported by the AXA Research Fund and the CSIC Intramural grant No. 201440I017 to G.R., the Spanish Ministry of Education, Culture and Sports FPU fellowship AP2012-3751 to E.M., the Spanish Ministry of Economy and Competitiveness grants No. AGL2013-49919-EXP and No. BIO2011-26741 to J.-A.D. and No. BFU2011-24691 to J.F.P., and the European Union grant No. FP7-KBBE-613745 (Programming synthetic networks for bio-based production of value chemicals) to A.J.Rodrigo Tarrega, G.; Majer, E.; Prakash, S.; Daros Arnau, JA.; Jaramillo, A.; Poyatos, JF. (2015). Exploring the Dynamics and Mutational Landscape of Riboregulation with a Minimal Synthetic Circuit in Living Cells. Biophysical Journal. 109(5):1070-1076. https://doi.org/10.1016/j.bpj.2015.07.021S10701076109

Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression

[EN] Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA-RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.EVOPROG [FP7-ICT-610730]; PROMYS [FP7-KBBE-613745 to A.J.]; Ministerio de Economia y Competitividad, Spain [BIO2011-26741 to J.-A.D.]; PRES Paris Sud grant (S.S.); EMBO long-term fellowship co-funded by Marie Curie actions [ALTF-1177-2011 A.J., G.R.]; AXA research fund; Ministerio de Educacion, Cultura y Deporte, Spain [AP2012-3751 to E.M.]. Funding for open access charge: EVOPROG [FP7-ICT-610730]; PROMYS [FP7-KBBE-613745].Shen, S.; Rodrigo Tarrega, G.; Prakash, S.; Majer, E.; Landrain, T.; Kirov, B.; Daros Arnau, JA.... (2015). Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression. Nucleic Acids Research. 43(10):5158-5170. https://doi.org/10.1093/nar/gkv287S515851704310Ulrich, L. E., Koonin, E. V., & Zhulin, I. B. (2005). One-component systems dominate signal transduction in prokaryotes. 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A new frontier in synthetic biology: automated design of small RNA devices in bacteria. Trends in Genetics, 29(9), 529-536. doi:10.1016/j.tig.2013.06.005Callura, J. M., Dwyer, D. J., Isaacs, F. J., Cantor, C. R., & Collins, J. J. (2010). Tracking, tuning, and terminating microbial physiology using synthetic riboregulators. Proceedings of the National Academy of Sciences, 107(36), 15898-15903. doi:10.1073/pnas.1009747107Callura, J. M., Cantor, C. R., & Collins, J. J. (2012). Genetic switchboard for synthetic biology applications. Proceedings of the National Academy of Sciences, 109(15), 5850-5855. doi:10.1073/pnas.1203808109Werstuck, G. (1998). Controlling Gene Expression in Living Cells Through Small Molecule-RNA Interactions. Science, 282(5387), 296-298. doi:10.1126/science.282.5387.296Wieland, M., & Hartig, J. S. (2008). Improved Aptazyme Design and In Vivo Screening Enable Riboswitching in Bacteria. 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Full design automation of multi-state RNA devices to program gene expression using energy-based optimization

[EN] Small RNAs (sRNAs) can operate as regulatory agents to control protein expression by interaction with the 59 untranslated region of the mRNA. We have developed a physicochemical framework, relying on base pair interaction energies, to design multi-state sRNA devices by solving an optimization problem with an objective function accounting for the stability of the transition and final intermolecular states. Contrary to the analysis of the reaction kinetics of an ensemble of sRNAs, we solve the inverse problem of finding sequences satisfying targeted reactions. We show here that our objective function correlates well with measured riboregulatory activity of a set of mutants. This has enabled the application of the methodology for an extended design of RNA devices with specified behavior, assuming different molecular interaction models based on Watson-Crick interaction. We designed several YES, NOT, AND, and OR logic gates, including the design of combinatorial riboregulators. In sum, our de novo approach provides a new paradigm in synthetic biology to design molecular interaction mechanisms facilitating future high-throughput functional sRNA design.Work supported by the grants FP7-ICT-043338 (BACTOCOM) to AJ, and BIO2011-26741 (Ministerio de Economia y Competitividad, Spain) to JAD. GR is supported by an EMBO long-term fellowship co-funded by Marie Curie actions (ALTF-1177-2011), and TEL by a PhD fellowship from the AXA Research Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Rodrigo Tarrega, G.; Landrain, TE.; Majer, E.; Daros Arnau, JA.; Jaramillo, A. (2013). Full design automation of multi-state RNA devices to program gene expression using energy-based optimization. PLoS Computational Biology. 9(8):1003172-1003172. https://doi.org/10.1371/journal.pcbi.1003172S1003172100317298Isaacs, F. J., Dwyer, D. J., & Collins, J. J. (2006). RNA synthetic biology. 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