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Open accessJournal ArticleDOI: 10.1038/S41467-021-21716-0

A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors.

04 Mar 2021-Nature Communications (Springer Science and Business Media LLC)-Vol. 12, Iss: 1, pp 1437-1437
Abstract: Biosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Here, using aptamer-coupled ribozyme libraries and a ribozyme regeneration method, de novo rapid in vitro evolution of RNA biosensors (DRIVER) enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identify and validate biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors are applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors are also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.

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Open accessJournal ArticleDOI: 10.1016/J.COGSC.2021.100567
Abstract: Plant natural products have been used for centuries to treat human illnesses, as recreational substances, and as flavouring and colouring agents in our food. Nowadays commercial scale manufacturing of plant natural products using extraction from natural resources or chemical synthesis pose challenges for environmental sustainability, such as species overexploitation. Bio-based synthesis of plant natural products in microbial cell factories can offer an attractive alternative as these processes reduce the use of natural plant resources and instead rely on renewable feedstocks as raw materials. Here, we review the most recent developments on sustainable supply of plant natural products by bio-based synthesis in yeast, with special focus on newly discovered and implemented plant natural product biosynthetic pathways, approaches for chemical diversification of natural products, and optimization of platform yeast cell factories. From this we discuss environmental considerations and the main challenges towards sustainable and robust microbial production of plant natural products based on fermentation.

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Open accessJournal ArticleDOI: 10.3390/PH14060554
Zachary J Tickner1, Michael Farzan1Institutions (1)
Abstract: Vectors developed from adeno-associated virus (AAV) are powerful tools for in vivo transgene delivery in both humans and animal models, and several AAV-delivered gene therapies are currently approved for clinical use. However, AAV-mediated gene therapy still faces several challenges, including limited vector packaging capacity and the need for a safe, effective method for controlling transgene expression during and after delivery. Riboswitches, RNA elements which control gene expression in response to ligand binding, are attractive candidates for regulating expression of AAV-delivered transgene therapeutics because of their small genomic footprints and non-immunogenicity compared to protein-based expression control systems. In addition, the ligand-sensing aptamer domains of many riboswitches can be exchanged in a modular fashion to allow regulation by a variety of small molecules, proteins, and oligonucleotides. Riboswitches have been used to regulate AAV-delivered transgene therapeutics in animal models, and recently developed screening and selection methods allow rapid isolation of riboswitches with novel ligands and improved performance in mammalian cells. This review discusses the advantages of riboswitches in the context of AAV-delivered gene therapy, the subsets of riboswitch mechanisms which have been shown to function in human cells and animal models, recent progress in riboswitch isolation and optimization, and several examples of AAV-delivered therapeutic systems which might be improved by riboswitch regulation.

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Topics: Riboswitch (54%)

Journal ArticleDOI: 10.1515/NTREV-2021-0099
01 Jan 2021-Nano Reviews
Abstract: Global overpopulation, industrial expansion, and urbanization have generated massive amounts of wastes. This is considered as a significant worldwide challenge that requires an urgent solution. Additionally, remarkable advances in the field of biomedicine have impacted the entire spectrum of healthcare and medicine. This has paved the way for further refining of the outcomes of biomedical strategies toward early detection and treatment of different diseases. Various nanomaterials (NMs) have been dedicated to different biomedical applications including drug delivery, vaccinations, imaging modalities, and biosensors. However, toxicity is still the main factor restricting their use. NMs recycled from different types of wastes present a pioneering approach to not only avoid hazardous effects on the environment, but to also implement circular economy practices, which are crucial to attain sustainable growth. Moreover, recycled NMs have been utilized as a safe, yet revolutionary alternative with outstanding potential for many biomedical applications. This review focuses on waste recycled NMs, their synthesis, properties, and their potential for multiple biomedical applications with special emphasis on their role in the early detection and control of multiple diseases. Their pivotal therapeutic actions as antimicrobial, anticancer, antioxidant nanodrugs, and vaccines will also be outlined. The ongoing advancements in the design of recycled NMs are expanding their diagnostic and therapeutic roles for diverse biomedical applications in the era of precision medicine.

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Open accessJournal ArticleDOI: 10.1016/J.CHEMBIOL.2021.10.013
Abstract: Conversion of in vitro selected aptamers into functional metabolic sensors is hampered by reduced in vivo aptamer binding and limited tunability of cellular metabolite levels. In this issue of Cell Chemical Biology, Ortega et al. (2021) construct RNA sensors of fructose-6-bisphosphate (FBP) that report on metabolite levels within single yeast cells.

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Topics: Aptamer (58%), Chemical biology (53%)

Open accessJournal ArticleDOI: 10.1097/CCM.0000000000005246
Manu Shankar-Hari, Hannah Wunsch1, Kathy Rowan, Mervyn Singer2  +2 moreInstitutions (3)
Topics: MEDLINE (59%)
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57 results found


Journal ArticleDOI: 10.1126/SCIENCE.2200121
Craig Tuerk1, Larry Gold1Institutions (1)
03 Aug 1990-Science
Abstract: High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.

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8,476 Citations


Journal ArticleDOI: 10.1038/346818A0
Andrew D. Ellington1, Jack W. Szostak1Institutions (1)
30 Aug 1990-Nature
Abstract: Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 10(10) random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.

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Topics: RNA (62%), Riboswitch (62%), Binding site (60%) ... read more

7,927 Citations


Open accessJournal ArticleDOI: 10.1093/BIOINFORMATICS/BTT593
01 Mar 2014-Bioinformatics
Abstract: Motivation The Illumina paired-end sequencing technology can generate reads from both ends of target DNA fragments, which can subsequently be merged to increase the overall read length. There already exist tools for merging these paired-end reads when the target fragments are equally long. However, when fragment lengths vary and, in particular, when either the fragment size is shorter than a single-end read, or longer than twice the size of a single-end read, most state-of-the-art mergers fail to generate reliable results. Therefore, a robust tool is needed to merge paired-end reads that exhibit varying overlap lengths because of varying target fragment lengths. Results We present the PEAR software for merging raw Illumina paired-end reads from target fragments of varying length. The program evaluates all possible paired-end read overlaps and does not require the target fragment size as input. It also implements a statistical test for minimizing false-positive results. Tests on simulated and empirical data show that PEAR consistently generates highly accurate merged paired-end reads. A highly optimized implementation allows for merging millions of paired-end reads within a few minutes on a standard desktop computer. On multi-core architectures, the parallel version of PEAR shows linear speedups compared with the sequential version of PEAR. Availability and implementation PEAR is implemented in C and uses POSIX threads. It is freely available at http://www.exelixis-lab.org/web/software/pear.

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2,626 Citations


Open accessJournal ArticleDOI: 10.1093/NAR/GKV437
Tilmann Weber1, Kai Blin1, Srikanth Duddela2, Daniel Krug2  +11 moreInstitutions (8)
Abstract: Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.

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1,575 Citations


Journal ArticleDOI: 10.1038/344467A0
Debra L. Robertson1, Gerald F. Joyce1Institutions (1)
29 Mar 1990-Nature
Abstract: The discovery of RNA enzymes has, for the first time, provided a single molecule that has both genetic and catalytic properties. We have devised techniques for the mutation, selection and amplification of catalytic RNA, all of which can be performed rapidly in vitro. Here we describe how these techniques can be integrated and performed repeatedly within a single reaction vessel. This allows evolution experiments to be carried out in response to artificially imposed selection constraints. We worked with the Tetrahymena ribozyme, a self-splicing group I intron derived from the large ribosomal RNA precursor of Tetrahymena thermophila that catalyses sequence-specific phosphoester transfer reactions involving RNA substrates. It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity. We selected for variant forms of the enzyme that could best react with a DNA substrate. This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme. The selected molecule represents the discovery of the first RNA enzyme known to cleave single-stranded DNA specifically.

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Topics: Ribozyme (63%), RNA (62%), RNA-dependent RNA polymerase (62%) ... read more

1,148 Citations


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YearCitations
20215