Open Access Publications

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Open access publications by faculty, postdocs, and graduate students in the Department of Chemical and Biomolecular Engineering

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A review of thermal and thermocatalytic valorization of food waste
(Green Chemistry, 2021-04-08) Ebikade, Elvis Osamudiamhen; Sadula, Sunitha; Gupta, Yagya; Vlachos, Dionisios G.
Food waste (FW) remains a global challenge due to the increasing demand for food production to support a growing global population and the lack of effective waste management technologies for recycling and upcycling. Unique compounds in FW – such as carbohydrates, proteins, lignin, fats, and extractives – can be repurposed to produce important biobased fuels, bulk chemicals, dietary supplements, adsorbents, and antibacterial products, among many others. We review the thermal and thermocatalytic FW valorization strategies and the fundamental pathways. We discuss the potential integration of various valorization processes, their economic viability, the technical and marketing challenges, and the need for further developments. By overcoming several technical hurdles, repurposing FW into modular plants can create exciting economic and environmental prospects.
Improved slit-shaped microseparator and its integration with a microreactor for modular biomanufacturing
(Green Chemistry, 2021-04-30) Bhattacharyya, Souryadeep; Desir, Pierre; Prodinger, Sebastian; Lobo, Raul F.; Vlachos, Dionisios G.
Modular and distributed biomanufacturing requires continuous flow microreactors integrated with efficient separation units operating at comparable time scales: biphasic reactive extraction of 5-hydroxymethyl furfural (HMF) by fructose dehydration is an excellent example. The liquid–liquid extraction (LLE) and fast reaction kinetics in biphasic microchannels can immensely benefit from a downstream microseparator enabling separation of an HMF-rich organic extract and an aqueous raffinate. Here we demonstrate the successful implementation of an effective slit-shaped microseparator for eleven organic-water biphasic systems. The microseparator successfully separates six of these over reasonable flow rates. The ratio of capillary and hydraulic pressures qualitatively rationalizes the separation performance, while a transition to non-segmented flow patterns correlates with performance deterioration. Acids and salts, integral parts of the chemistry, significantly expand the flow rates for efficient separation enabling a broader slate of organic solvents. For the MIBK/water biphasic system, we demonstrate perfect separation performance over a 16-fold variation in the organic to aqueous flow ratio. Here we also integrate the microseparator and extractive microreactor into a modular system and achieve an HMF yield of up to 93% – the highest reported fractional HMF productivity of 27.9 min−1 – at an ultrashort residence time of 2 s. This unprecedented performance is maintained over a 50-fold fructose concentration range and is stable with time-on-stream. This microseparator exhibits a ten-fold reduction in separation time and substantial energy savings over conventional decanters. As such, it holds promise for continuous process intensification and modular biomanufacturing.
Estrogenic activity of lignin-derivable alternatives to bisphenol A assessed via molecular docking simulations
(RSC Advances, 2021-06-23) Amitrano, Alice; Mahajan, Jignesh S.; Korley, LaShanda T. J.; Epps, Thomas H. III
Lignin-derivable bisphenols are potential alternatives to bisphenol A (BPA), a suspected endocrine disruptor; however, a greater understanding of structure–activity relationships (SARs) associated with such lignin-derivable building blocks is necessary to move replacement efforts forward. This study focuses on the prediction of bisphenol estrogenic activity (EA) to inform the design of potentially safer BPA alternatives. To achieve this goal, the binding affinities to estrogen receptor alpha (ERα) of lignin-derivable bisphenols were calculated via molecular docking simulations and correlated to median effective concentration (EC50) values using an empirical correlation curve created from known EC50 values and binding affinities of commercial (bis)phenols. Based on the correlation curve, lignin-derivable bisphenols with binding affinities weaker than ∼−6.0 kcal mol−1 were expected to exhibit no EA, and further analysis suggested that having two methoxy groups on an aromatic ring of the bio-derivable bisphenol was largely responsible for the reduction in binding to ERα. Such dimethoxy aromatics are readily sourced from the depolymerization of hardwood biomass. Additionally, bulkier substituents on the bridging carbon of lignin-bisphenols, like diethyl or dimethoxy, were shown to weaken binding to ERα. And, as the bio-derivable aromatics maintain major structural similarities to BPA, the resultant polymeric materials should possess comparable/equivalent thermal (e.g., glass transition temperatures, thermal decomposition temperatures) and mechanical (e.g., tensile strength, modulus) properties to those of polymers derived from BPA. Hence, the SARs established in this work can facilitate the development of sustainable polymers that maintain the performance of existing BPA-based materials while simultaneously reducing estrogenic potential.
Self-assembling protein nanocages for modular enzyme assembly by orthogonal bioconjugation
(Biotechnology Progress, 2021-06-25) Berckman, Emily A.; Chen, Wilfred
The wide variety of enzymatic pathways that can benefit from enzyme scaffolding is astronomical. While enzyme co-localization based on protein, DNA, and RNA scaffolds has been reported, we still lack scaffolds that offer well-defined and uniform three-dimensional structures for enzyme organization. Here we reported a new approach for protein co-localization using naturally occurring protein nanocages as a scaffold. Two different nanocages, the 25 nm E2 and the 34 nm heptatitis B virus, were used to demonstrate the successfully co-localization of the endoglucanase CelA and cellulose binding domain using the robust SpyTag/SpyCatcher bioconjugation chemistry. Because of the simplicity of the assembly, this strategy is useful not only for in vivo enzyme cascading but also the potential for in vivo applications as well.
Hydrogel nanoparticle degradation influences the activation and survival of primary macrophages
(Journal of Materials Chemistry B, 2021-06-28) Jarai, Bader M.; Stillman, Zachary; Fromen, Catherine A.
The effect of nanoparticle (NP) internalization on cell fate has emerged as an important consideration for nanomedicine design, as macrophages and other phagocytes are the primary clearance mechanisms of administered NP formulations. Pro-survival signaling is thought to be concurrent with phagocytosis and recent work has shown increased macrophage survival following lysosomal processing of internalized NPs. These observations have opened the door to explorations of NP physiochemical properties aimed at tuning the NP-driven macrophage survival at the lysosomal synapse. Here, we report that NP-induced macrophage survival and activation is strongly dependent on NP degradation rate using a series of thiol-containing poly(ethylene glycol) diacrylate-based NPs of equivalent size and zeta potential. Rapidly degrading, high thiol-containing NPs allowed for dramatic enhancement of cell longevity that was concurrent with macrophage stimulation after 2 weeks in ex vivo culture. While equivalent NP internalization resulted in suppressed caspase activity across the NP series, macrophage activation was correlated with increasing thiol content, leading to increased lysosomal activity and a robust pro-survival phenotype. Our results provide insight on tuning NP physiochemical properties as design handles for maximizing ex vivo macrophage longevity, which has implications for improving macrophage-based immune assays, biomanufacturing, and cell therapies.
The impact of differential lignin S/G ratios on mutagenicity and chicken embryonic toxicity
(Journal of Applied Toxicology, 2021-08-27) Zhang, Xinwen; Levia, Delphis F.; Ebikade, Elvis Osamudiamhen; Chang, Jeffrey; Vlachos, Dionisios G.; Wu, Changqing
Lignin and lignin-based materials have received considerable attention in various fields due to their promise as sustainable feedstocks. Guaiacol (G) and syringol (S) are two primary monolignols that occur in different ratios for different plant species. As methoxyphenols, G and S have been targeted as atmospheric pollutants and their acute toxicity examined. However, there is a rare understanding of the toxicological properties on other endpoints and mixture effects of these monolignols. To fill this knowledge gap, our study investigated the impact of different S/G ratios (0.5, 1, and 2) and three lignin depolymerization samples from poplar, pine, and miscanthus species on mutagenicity and developmental toxicity. A multitiered method consisted of in silico simulation, in vitro Ames test, and in vivo chicken embryonic assay was employed. In the Ames test, syringol showed a sign of mutagenicity, whereas guaiacol did not, which agreed with the T.E.S.T. simulation. For three S and G mixture and lignin monomers, mutagenic activity was related to the proportion of syringol. In addition, both S and G showed developmental toxicity in the chicken embryonic assay and T.E.S.T. simulation, and guaiacol had a severe effect on lipid peroxidation. A similar trend and comparable developmental toxicity levels were detected for S and G mixtures and the three lignin depolymerized monomers. This study provides data and insights on the differential toxicity of varying S/G ratios for some important building blocks for bio-based materials.
Scalable 3D-printed lattices for pressure control in fluid applications
(AIChE Journal, 2021-09-23) Woodward, Ian R.; Attia, Lucas; Patel, Premal; Fromen, Catherine A.
Additive manufacturing affords precise control over geometries with high degrees of complexity and predefined structure. Lattices are one class of additive-only structures which have great potential in directing transport phenomena because they are highly ordered, scalable, and modular. However, a comprehensive description of how these structures scale and interact in heterogeneous systems is still undetermined. To advance this aim, we designed cubic and Kelvin lattices at two sub-5-mm length scales and compared published correlations to the experimental pressure gradient in pipes ranging from 12 to 52 mm diameter. We further investigated all combinations of the four lattices to evaluate segmented combinatorial behavior. The results suggest that a single correlation can describe pressure behavior for different lattice geometries and scales. Furthermore, combining lattice systems in series has a complex effect that is sensitive to part geometry. Together, these developments support the promise for tailored, modular lattice systems at laboratory scales and beyond.
Outer Membrane Vesicles (OMVs) Enabled Bio-applications: A critical review
(Biotechnology and Bioengineering, 2021-10-26) Huang, Yikun; Nieh, Mu-Ping; Chen, Wilfred; Lei, Yu
Outer membrane vesicles (OMVs) are nanoscale spherical vesicles released from Gram-negative bacteria. The lipid bilayer membrane structure of OMVs consists of similar components as bacterial membrane and thus has attracted more and more attention in exploiting OMVs' bio-applications. Although the endotoxic lipopolysaccharide on natural OMVs may impose potential limits on their clinical applications, genetic modification can reduce their endotoxicity and decorate OMVs with multiple functional proteins. These genetically engineered OMVs have been employed in various fields including vaccination, drug delivery, cancer therapy, bioimaging, biosensing, and enzyme carrier. This review will first briefly introduce the background of OMVs followed by recent advances in functionalization and various applications of engineered OMVs with an emphasis on the working principles and their performance, and then discuss about the future trends of OMVs in biomedical applications.
Nanocrystalline protein domains via salting-out
(Acta Crystallographica Section F: Structural Biology Communications, 2021-11-02) Greene, D. G.; Modla, S.; Sandler, S. I.; Wagner, N. J.; Lenhoff, A. M.
Protein salting-out is a well established phenomenon that in many cases leads to amorphous structures and protein gels, which are usually not considered to be useful for protein structure determination. Here, microstructural measurements of several different salted-out protein dense phases are reported, including of lysozyme, ribonuclease A and an IgG1, showing that salted-out protein gels unexpectedly contain highly ordered protein nanostructures that assemble hierarchically to create the gel. The nanocrystalline domains are approximately 10–100 nm in size, are shown to have structures commensurate with those of bulk crystals and grow on time scales in the order of an hour to a day. Beyond revealing the rich, hierarchical nanoscale to mesoscale structure of protein gels, the nanocrystals that these phases contain are candidates for structural biology on next-generation X-ray free-electron lasers, which may enable the study of biological macromolecules that are difficult or impossible to crystallize in bulk.
Polyolefin plastic waste hydroconversion to fuels, lubricants, and waxes: a comparative study
(Reaction Chemistry and Engineering, 2021-12-01) Kots, Pavel A.; Vance, Brandon C.; Vlachos, Dionisios G.
Hydroconversion technologies have surged to the forefront of deconstructing plastic waste. Recent studies have been performed over several catalysts with varying conditions and plastics that make comparisons difficult. We compile and compare data from the literature by introducing various metrics and perform a simple energy analysis. We draw mechanistic similarities to and differences from the past literature on small alkane hydroconversion and leverage the former to propose standard approaches to tune product selectivity. We exemplify the plastics materials gap and the challenges it creates. Finally, we discuss the current limitations and suggest future work.
Comparison of lunar and Martian regolith simulant-based geopolymer cements formed by alkali-activation for in-situ resource utilization
(Advances in Space Research, 2021-12-20) Mills, Jennifer N.; Katzarova, Maria; Wagner, Norman J.
Future human space exploration and habitation on the lunar and Martian surfaces necessitates in-situ resource utilization (ISRU) for the development of construction materials tailored for infrastructure and environmental protection. Here we explore the use of lunar and Martian regoliths to create construction materials with properties suitable for such structures as landing pads. Alkali activation of a spectrum of lunar and Martian regolith simulants generates geopolymer binders under ambient and vacuum curing conditions as well as exposure to extreme high and low temperatures (600 and −80 °C). Compressive strength is reduced for binders prepared from each simulant after curing under vacuum and exposure to sub-zero temperatures. In lunar simulant binders, the compressive strength is increased after heating to 600 °C, but the opposite effect is observed in the Martian simulant binder. Amorphous aluminosilicate content and percentage of small particles in the simulants are hypothesized to have a positive impact on compressive strength under ambient curing. Iron and magnesium content may be responsible for decreased compressive strength of the Martian binder after heating. This study offers a robust framework for comparing performance of different simulants under the same curing protocols and environmental exposures, as well as offering insight as to the effects of vacuum curing, and exposure to high and low temperature environments on cured binder samples. Developing a landing pad by transporting activator to the lunar surface is shown to be conceptually feasible within current payload constraints.
Accelerating manufacturing for biomass conversion via integrated process and bench digitalization: a perspective
(Reaction Chemistry and Engineering, 2022-01-25) Batchu, Sai Praneet; Hernandez, Borja; Malhotra, Abhinav; Fang, Hui; Ierapetritou, Marianthi; Vlachos, Dionisios G.
We present a perspective for accelerating biomass manufacturing via digitalization. We summarize the challenges for manufacturing and identify areas where digitalization can help. A profound potential in using lignocellulosic biomass and renewable feedstocks, in general, is to produce new molecules and products with unmatched properties that have no analog in traditional refineries. Discovering such performance-advantaged molecules and the paths and processes to make them rapidly and systematically can transform manufacturing practices. We discuss retrosynthetic approaches, text mining, natural language processing, and modern machine learning methods to enable digitalization. Laboratory and multiscale computation automation via active learning are crucial to complement existing literature and expedite discovery and valuable data collection without a human in the loop. Such data can help process simulation and optimization select the most promising processes and molecules according to economic, environmental, and societal metrics. We propose the close integration between bench and process scale models and data to exploit the low dimensionality of the data and transform the manufacturing for renewable feedstocks.
Extracellular vesicles facilitate large-scale dynamic exchange of proteins and RNA among cultured Chinese hamster ovary and human cells
(Biotechnology and Bioengineering, 2022-02-04) Belliveau, Jessica; Papoutsakis, Eleftherios T.
Cells in culture are viewed as unique individuals in a large population communicating through extracellular molecules and, more recently extracellular vesicles (EVs). Our data here paint a different picture: large-scale exchange of cellular material through EVs. To visualize the dynamic production and cellular uptake of EVs, we used correlative confocal microscopy and scanning electron microscopy, as well as flow cytometry to interrogate labeled cells. Using cells expressing fluorescent proteins (GFP, miRFP703) and cells tagged with protein and RNA dyes, we show that Chinese hamster ovary (CHO) cells dynamically produce and uptake EVs to exchange proteins and RNAs at a large scale. Applying a simple model to our data, we estimate, for the first time, the per cell-specific rates of EV production (68 and 203 microparticles and exosomes, respectively, per day). This EV-mediated massive exchange of cellular material observed in CHO cultures was also observed in cultured human CHRF-288-11 and primary hematopoietic stem and progenitor cells. This study demonstrates an underappreciated massive protein and RNA exchange between cells mediated by EVs spanning cell type, suggesting that the proximity of cells in normal and tumor tissues may also result in prolific exchange of cellular material. This exchange would be expected to homogenize the cell-population cytosol and dynamically regulate cell proliferation and the cellular state.
Nanoparticle Internalization Promotes the Survival of Primary Macrophages
(Wiley-VCH GmbH, 2022-02-09) Jarai, Bader M.; Fromen, Catherine A.; Bader M. Jarai, Catherine A. Fromen; Catherine A. Fromen; Bader M. Jarai
Macrophages, a class of tissue resident innate immune cells, are responsible forsequestering foreign objects through the process of phagocytosis, making them apromising target for immune modulation via particulate engineering. Herein, it isreported that nanoparticle (NP) dosing and cellular internalization via phago-cytosis significantly enhance survival of ex vivo cultures of primary bone marrow-derived, alveolar, and peritoneal macrophages over particle-free controls. Theenhanced survival is attributed to suppression of caspase-dependent apoptosisand is linked to phagocytosis and lysosomal signaling. Uniquely, poly(ethyleneglycol)-based NP treatment extends cell viability in the absence of macrophagepolarization and enhances expression of prosurvival B cell lymphoma-2 (Bcl-2)protein in macrophages following multiple routes of in vivo administration. Theenhanced survival phenomenon is also applicable to NPs of alternative chem-istries, indicating the potential universality of this phenomenon with relevantdrug delivery particles. Thesefindings provide a framework for extending thelifespan of primary macrophages ex vivo for drug screening, vaccine studies,and cell therapies and have implications for in vivo particulate immune-engineering applications.
Area-based Image Analysis Algorithm for Quantification of Macrophage-fibroblast Cocultures
(Journal of Visualized Experiments, 2022-02-15) Borjigin, Tohn; Boddupalli, Anuraag; Sullivan, Millicent O.
Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either fluorescence detection approaches, such as immunofluorescent staining or transfection with fluorescent proteins or edge detection techniques, which are often error-prone due to noise and other non-idealities in the image background. We designed a new algorithm that could accurately count and distinguish macrophages and fibroblasts, cells of different phenotypes that often colocalize during tissue regeneration. MATLAB was used to implement the algorithm, which differentiated distinct cell types based on differences in height from the background. A primary algorithm was developed using an area-based method to account for variations in cell size/structure and high-density seeding conditions. Non-idealities in cell structures were accounted for with a secondary, iterative algorithm utilizing internal parameters such as cell coverage computed using experimental data for a given cell type. Finally, an analysis of coculture environments was carried out using an isolation algorithm in which various cell types were selectively excluded based on the evaluation of relative height differences within the image. This approach was found to accurately count cells within a 5% error margin for monocultured cells and within a 10% error margin for cocultured cells.
Perspective—Trends in the Recognition of Women in Electrochemistry
(Journal of The Electrochemical Society, 2022-02-28) Oliveira, Alexandra M.; Beswick, Rebecca R.; Yan, Yushan
Like many science and engineering fields, electrochemistry has historically been dominated by male researchers. This perspective celebrates the contributions of female electrochemists and studies trends in the number of women recognized by the International Society of Electrochemistry (ISE), the Electrochemical Society (ECS), the National Academy of Engineering (NAE), and the National Academy of Sciences (NAS) for their work in electrochemical fields. In recent years, women are being recognized more frequently for impactful electrochemical research, signaling the beginning of a journey toward more equal representation in a field in which men and women together can solve the world's greatest energy challenges.
Impact of collagen-like peptide (CLP) heterotrimeric triple helix design on helical thermal stability and hierarchical assembly: a coarse-grained molecular dynamics simulation study
(Soft Matter, 2022-04-05) Taylor, Phillip A.; Kloxin, April M.; Jayaraman, Arthi
Collagen-like peptides (CLP) are multifunctional materials garnering a lot of recent interest from the biomaterials community due to their hierarchical assembly and tunable physicochemical properties. In this work, we present a computational study that links the design of CLP heterotrimers to the thermal stability of the triple helix and their self-assembly into fibrillar aggregates and percolated networks. Unlike homotrimeric helices, the CLP heterotrimeric triple helices in this study are made of CLP strands of different chain lengths that result in ‘sticky’ ends with available hydrogen bonding groups. These ‘sticky’ ends at one end or both ends of the CLP heterotrimer then facilitate inter-helix hydrogen bonding leading to self-assembly into fibrils (clusters) and percolated networks. We consider the cases of three sticky end lengths – two, four, and six repeat units – present entirely on one end or split between two ends of the CLP heterotrimer. We observe in CLP heterotrimer melting curves generated using coarse grained Langevin dynamics simulations at low CLP concentration that increasing sticky end length results in lower melting temperatures for both one and two sticky ended CLP designs. At higher CLP concentrations, we observe non-monotonic trends in cluster sizes with increasing sticky end length with one sticky end but not for two sticky ends with the same number of available hydrogen bonding groups as the one sticky end; this nonmonotonicity stems from the formation of turn structures stabilized by hydrogen bonds at the single, sticky end for sticky end lengths greater than four repeat units. With increasing CLP concentration, heterotrimers also form percolated networks with increasing sticky end length with a minimum sticky end length of four repeat units required to observe percolation. Overall, this work informs the design of thermoresponsive, peptide-based biomaterials with desired morphologies using strand length and dispersity as a handle for tuning thermal stability and formation of supramolecular structures.
Dynamic modulation of enzyme activity by synthetic CRISPR–Cas6 endonucleases
(Nature Chemical Biology, 2022-04-25) Mitkas, Alexander A.; Valverde, Mauricio; Chen, Wilfred
In nature, dynamic interactions between enzymes play a crucial role in defining cellular metabolism. By controlling the spatial and temporal organization of these supramolecular complexes called metabolons, natural metabolism can be tuned in a highly dynamic manner. Here, we repurpose the CRISPR–Cas6 family proteins as a synthetic strategy to create dynamic metabolons by combining the ease of RNA processing and the predictability of RNA hybridization for protein assembly. By disturbing RNA–RNA networks using toehold-mediated strand displacement reactions, on-demand assembly and disassembly are achieved using both synthetic RNA triggers and mCherry messenger RNA. Both direct and ‘Turn-On’ assembly of the pathway enzymes tryptophan-2-monooxygenase and indoleacetamide hydrolase can enhance indole-3-acetic acid production by up to ninefold. Even multimeric enzymes can be assembled to improve malate production by threefold. By interfacing with endogenous mRNAs, more complex metabolons may be constructed, resulting in a self-responsive metabolic machinery capable of adapting to changing cellular demand.
Design and qualification of a bench-scale model for municipal waste-to-energy combustion
(Journal of the Air & Waste Management Association, 2022-04-25) Giraud, Robert J.; Taylor, Philip H.; Diemer, R. Bertrum; Huang, Chin-Pao
This paper reports the design and qualification of the first purpose-built, bench-scale reactor system to model the municipal waste-to-energy combustion of fluorinated polymers. Using the principle of similarity, the gas-phase combustion zone of a typical municipal waste-to-energy plant has been scaled down to the bench with a focus on chemical similarity. Chemical similarity is achieved in large part through the use of methanol as a surrogate for municipal solid waste (MSW). Review of prior research shows that methanol is one of the major volatile products expected during MSW thermal conversion in the fuel bed of waste-to-energy plants. Like full-scale waste-energy plants, the design of the bench-scale model includes a flame zone and a post-flame zone. Maintaining steady methanol vapor flow premixed with air to the model reactor system ensures stable combustion resulting in bench-scale CO emission levels comparable to those of full-scale waste-to-energy plants. Since investigation of fluorinated polymer combustion includes trace analysis of exhaust gas for perfluorooctanoic acid (PFOA), qualification testing focused on PFOA collection efficiency. High PFOA collection efficiency (>90%) demonstrated the capability of the reactor system in transporting and absorbing PFOA that may be generated during high-temperature combustion testing of fluorinated polymers. Overall, the bench-scale system is qualified for its intended use to investigate potential generation of PFOA from combustion of fluorinated polymers under conditions representative of waste-to-energy combustion. Implications: Decision makers depend on environmental researchers to provide reliable predictions of pollutant emissions from waste combustion of polymers at end of product life. Reliable predictions are especially important with regard to questions about potential PFOA emissions from municipal waste combustion of fluorinated polymers. Results from qualification testing confirm that the novel bench-scale model reactor system is capable of representing gas-phase municipal waste combustion behavior upstream of air pollution control and generating representative exhaust gas samples for off-line trace-level analysis of PFOA.
A microRNA-gated thgRNA platform for multiplexed activation of gene expression in mammalian cells
(Chemical Communications, 2022-04-26) Hunt, Victoria M.; Chen, Wilfred
To effectively reprogram cellular regulatory networks towards desired phenotypes, it is critical to have the ability to provide precise gene regulation in a spatiotemporal manner. We have previously engineered toehold-gated guide RNA (thgRNA) to enable conditional activation of dCas9-mediated transcriptional upregulation in mammalian cells using synthetic RNA triggers. Here, we demonstrate that microRNA (miR)-gated thgRNAs can be transcribed by type II RNA polymerase to allow multiplexed transcriptional activation using both mRNA and miR. Activation is achieved only by proper miR-mediated processing of the flanking 5′ cap and 3′ poly A tail and hairpin unblocking by mRNA via strand displacement. This new AND-gate design is exploited to elicit conditional protein degradation based on induced expression of a specific ubiquibody. This new strategy may find many new applications in an RNA-responsive manner.

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