81 results on '"Peter J. Unrau"'
Search Results
2. High-throughput detection of RNA processing in bacteria
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Erin E. Gill, Luisa S. Chan, Geoffrey L. Winsor, Neil Dobson, Raymond Lo, Shannan J. Ho Sui, Bhavjinder K. Dhillon, Patrick K. Taylor, Raunak Shrestha, Cory Spencer, Robert E. W. Hancock, Peter J. Unrau, and Fiona S. L. Brinkman
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RNA processing ,Nucleases ,Transcription ,RNA-Seq ,Gene expression ,Gene regulation ,Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Understanding the RNA processing of an organism’s transcriptome is an essential but challenging step in understanding its biology. Here we investigate with unprecedented detail the transcriptome of Pseudomonas aeruginosa PAO1, a medically important and innately multi-drug resistant bacterium. We systematically mapped RNA cleavage and dephosphorylation sites that result in 5′-monophosphate terminated RNA (pRNA) using monophosphate RNA-Seq (pRNA-Seq). Transcriptional start sites (TSS) were also mapped using differential RNA-Seq (dRNA-Seq) and both datasets were compared to conventional RNA-Seq performed in a variety of growth conditions. Results The pRNA-Seq library revealed known tRNA, rRNA and transfer-messenger RNA (tmRNA) processing sites, together with previously uncharacterized RNA cleavage events that were found disproportionately near the 5′ ends of transcripts associated with basic bacterial functions such as oxidative phosphorylation and purine metabolism. The majority (97%) of the processed mRNAs were cleaved at precise codon positions within defined sequence motifs indicative of distinct endonucleolytic activities. The most abundant of these motifs corresponded closely to an E. coli RNase E site previously established in vitro. Using the dRNA-Seq library, we performed an operon analysis and predicted 3159 potential TSS. A correlation analysis uncovered 105 antiparallel pairs of TSS that were separated by 18 bp from each other and were centered on single palindromic TAT(A/T)ATA motifs (likely − 10 promoter elements), suggesting that, consistent with previous in vitro experimentation, these sites can initiate transcription bi-directionally and may thus provide a novel form of transcriptional regulation. TSS and RNA-Seq analysis allowed us to confirm expression of small non-coding RNAs (ncRNAs), many of which are differentially expressed in swarming and biofilm formation conditions. Conclusions This study uses pRNA-Seq, a method that provides a genome-wide survey of RNA processing, to study the bacterium Pseudomonas aeruginosa and discover extensive transcript processing not previously appreciated. We have also gained novel insight into RNA maturation and turnover as well as a potential novel form of transcription regulation. NOTE: All sequence data has been submitted to the NCBI sequence read archive. Accession numbers are as follows: [NCBI sequence read archive: SRX156386, SRX157659, SRX157660, SRX157661, SRX157683 and SRX158075]. The sequence data is viewable using Jbrowse on www.pseudomonas.com.
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- 2018
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3. Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells
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Alexis Autour, Sunny C. Y. Jeng, Adam D. Cawte, Amir Abdolahzadeh, Angela Galli, Shanker S. S. Panchapakesan, David Rueda, Michael Ryckelynck, and Peter J. Unrau
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Science - Abstract
Many aptamer-fluorophore complexes suffer from low quantum yield and low extinction coefficients limiting their usability. Here the authors isolate new Mango aptamers with improved fluorescent properties for both fixed and live-cell imaging by using competitive ligand binding microfluidic selection.
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- 2018
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4. Purification of RNA Mango Tagged Native RNA-protein Complexes from Cellular Extracts Using TO1-Desthiobiotin Fluorophore Ligand
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Shanker Panchapakesan, Sunny Jeng, and Peter J. Unrau
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Biology (General) ,QH301-705.5 - Abstract
A native purification strategy using RNA Mango for RNA based purification of RNA-protein complexes is described. The RNA Mango aptamer is first genetically engineered into the RNA of interest. RNA Mango containing complexes obtained from cleared cellular native extracts are then immobilized onto TO1-Desthiobiotin saturated streptavidin agarose beads. The beads are washed to remove non-specific complexes and then the RNA Mango containing complexes are eluted by the addition of free biotin to the beads. Since the eluted complexes are native and fluorescent, a second purification step such as size exclusion chromatography can easily be added and the purified complexes tracked by monitoring fluorescence. The high purity native complexes resulting from this two-step purification strategy can be then used for further biochemical characterization.
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- 2018
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5. RNA Synthesis by in Vitro Selected Ribozymes for Recreating an RNA World
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Lyssa L. Martin, Peter J. Unrau, and Ulrich F. Müller
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RNA world ,ribozyme ,origin of life ,Science - Abstract
The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst. This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms. This review focuses on three types of ribozymes that could have been involved in the synthesis of RNA, the core activity in the self-replication of RNA world organisms. These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates. The strengths and weaknesses regarding each ribozyme’s possible function in a self-replicating RNA network are described, together with the obstacles that need to be overcome before an RNA world organism can be generated in the laboratory.
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- 2015
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6. Purification of Histidine-Tagged T4 RNA Ligase from E. coli
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Qing S. Wang and Peter J. Unrau
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Biology (General) ,QH301-705.5 - Abstract
Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1–3 mg pure protein/g cell pellet. A simple purification procedure ensures that the enzyme is de-adenylated to levels comparable to those found for many commercial preparations. The purified protein has very low levels of RNase contamination and functioned normally in a variety of activity assays.
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- 2002
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7. Phosphoryl Transfer Ribozymes
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Peter J. Unrau and Razvan Cojocaru
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chemistry.chemical_classification ,RNA world hypothesis ,DNA ligase ,Enzyme ,chemistry ,biology ,Kinase ,Stereochemistry ,biology.protein ,Ribozyme ,Glycosidic bond ,Polymerase - Published
- 2021
8. Microchip RT-PCR Detection of Nasopharyngeal SARS-CoV-2 Samples
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Razvan Cojocaru, Iqra M. Yaseen, Gordon Ritchie, Maxim Slyadnev, Sikander Gill, Don D. Sin, Marc G. Romney, Christopher F. Lowe, and Peter J. Unrau
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0301 basic medicine ,Canada ,Coronavirus disease 2019 (COVID-19) ,Point-of-care testing ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Pcr assay ,Real-Time Polymerase Chain Reaction ,Article ,Pathology and Forensic Medicine ,03 medical and health sciences ,0302 clinical medicine ,Limit of Detection ,Lab-On-A-Chip Devices ,Nasopharynx ,Humans ,Medicine ,Detection limit ,SARS-CoV-2 ,business.industry ,COVID-19 ,Diagnostic test ,Gold standard (test) ,Virology ,3. Good health ,Benchmarking ,030104 developmental biology ,Real-time polymerase chain reaction ,Point-of-Care Testing ,COVID-19 Nucleic Acid Testing ,030220 oncology & carcinogenesis ,RNA, Viral ,Molecular Medicine ,Reagent Kits, Diagnostic ,business - Abstract
Fast, accurate, and reliable diagnostic tests are critical for controlling the spread of the coronavirus disease 2019 (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The current gold standard for testing is real-time PCR; however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 minutes), user-friendly, and accurate microchip real-time PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. Microchips preloaded with COVID-19 primers and probes for the N gene accommodate 1.2-μL reaction volumes, lowering the required reagents by 10-fold compared with tube-based real-time PCR. We validated our assay using contrived reference samples and 21 clinical samples from patients in Canada, determining a limit of detection of 1 copy per reaction. The microchip real-time PCR provides a significantly lower resource alternative to the Centers for Disease Control and Prevention-approved real-time RT-PCR assays with comparable sensitivity, showing 100% positive and negative predictive agreement of clinical samples.
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- 2021
9. RNA Secondary Structure Prediction Via Energy Density Minimization.
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Can Alkan, Emre Karakoç, Süleyman Cenk Sahinalp, Peter J. Unrau, H. Alexander Ebhardt, Kaizhong Zhang, and Jeremy Buhler
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- 2006
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10. Single-molecule RNA Imaging Using Mango II Arrays
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Adam D. Cawte, Haruki Iino, Peter J. Unrau, David Rueda, and Medical Research Council
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Mangifera ,Fluorogenic RNA Aptamer ,Chemistry ,RNA ,food and beverages ,Folding (DSP implementation) ,Superresolution microscopy ,Aptamers, Nucleotide ,0601 Biochemistry and Cell Biology ,Fluorescence ,Fluorescence intensity ,RNA Aptamers ,Single Molecule Microscopy ,Single-Molecule Microscopy ,Biochemistry ,Live cell imaging ,Spinacia oleracea ,RNA-Mango ,0399 Other Chemical Sciences ,Molecule ,RNA dynamics ,Fluorescent Dyes ,Developmental Biology - Abstract
In recent years, fluorogenic RNA aptamers, such as Spinach, Broccoli, Corn, Mango, Coral, and Pepper have gathered traction as an efficient alternative labeling strategy for background-free imaging of cellular RNAs. However, their application has been somewhat limited by relatively inefficient folding and fluorescent stability. With the recent advent of novel RNA-Mango variants which are improved in both fluorescence intensity and folding stability in tandem arrays, it is now possible to image RNAs with single-molecule sensitivity. Here we discuss the protocol for imaging Mango II tagged RNAs in both fixed and live cells.
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- 2021
11. Single-Molecule RNA Imaging Using Mango II Arrays
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Adam D, Cawte, Haruki, Iino, Peter J, Unrau, and David S, Rueda
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Mangifera ,Spinacia oleracea ,RNA ,Aptamers, Nucleotide ,Fluorescent Dyes - Abstract
In recent years, fluorogenic RNA aptamers, such as Spinach, Broccoli, Corn, Mango, Coral, and Pepper have gathered traction as an efficient alternative labeling strategy for background-free imaging of cellular RNAs. However, their application has been somewhat limited by relatively inefficient folding and fluorescent stability. With the recent advent of novel RNA-Mango variants which are improved in both fluorescence intensity and folding stability in tandem arrays, it is now possible to image RNAs with single-molecule sensitivity. Here we discuss the protocol for imaging Mango II tagged RNAs in both fixed and live cells.
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- 2021
12. Decision letter: Witnessing the structural evolution of an RNA enzyme
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Peter J. Unrau and Donald Burke
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chemistry.chemical_classification ,Enzyme ,Chemistry ,RNA ,Computational biology ,Structural evolution - Published
- 2021
13. Fluorogenic aptamers for imaging and manipulation of cellular RNAs
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Kristen J. Kong, Xiaocen Lu, Elena Dolgosheina, Haruki Iino, Adam Cawte, David S. Rueda, and Peter J. Unrau
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Biophysics - Published
- 2022
14. Non-coding NFKBIZ 3′ UTR mutations promote cell growth and resistance to targeted therapeutics in diffuse large B-cell lymphoma
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Miguel Alcaide, Christopher Rushton, Laura K. Hilton, Adele Telenius, Anja Mottok, Jie Gao, Shannon Healy, Daniel J. Hodson, Nicole Thomas, Louis M. Staudt, Jeffrey Tang, Kostiantyn Dreval, Wyndham H. Wilson, Peter J. Unrau, Razvan Cojocaru, David Scott, Ryan D. Morin, Christian Steidl, and Sarah E. Arthur
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Untranslated region ,Mutation ,Three prime untranslated region ,Cancer research ,medicine ,Cancer ,Germinal center ,Biology ,medicine.disease ,medicine.disease_cause ,Gene ,Diffuse large B-cell lymphoma ,Lymphoma - Abstract
Amplifications and non-coding 3′ UTR mutations affecting NFKBIZ have been identified as recurrent genetic events in diffuse large B-cell lymphoma (DLBCL). We confirm the prevalence and pattern of NFKBIZ 3′ UTR mutations in independent cohorts and determine they are enriched in the ABC subtype as well as the recently described novel BN2/C1/NOTCH2 classes of DLBCL. Presently, the effects of and mechanism by which non-coding mutations can act as cancer drivers has been relatively unexplored. Here, we provide a functional characterization of these non-coding NFKBIZ 3′ UTR mutations. We demonstrate that the resulting elevated expression of IκB-ζ confers growth advantage in DLBCL cell lines and primary germinal center B-cells as well as nominate novel IκB-ζ target genes with potential therapeutic implications. The limited responses to targeted treatments in DLBCL, particularly those targeting the NF-κB axis, led us to investigate and confirm that NFKBIZ 3′ UTR mutations affect response to therapeutics and suggest it may be a useful predictive biomarker.Statement of SignificanceThrough functional characterization we reveal that non-coding NFKBIZ 3′ UTR mutations are a common driver in DLBCL, and mutation status may be a relevant biomarker to predict poor response to therapeutics targeting the NF-κB pathway.
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- 2021
15. RNA | Ribozymes and Evolution
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Razvan Cojocaru and Peter J. Unrau
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0303 health sciences ,03 medical and health sciences ,0302 clinical medicine ,030217 neurology & neurosurgery ,030304 developmental biology - Published
- 2021
16. RNA Mango: a versatile tool to track cellular RNAs and for rapid viral RNA detection
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David S. Rueda, Haruki Iino, Adam D. Cawte, and Peter J. Unrau
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Biophysics - Published
- 2022
17. RNA detection with high specificity and sensitivity using nested fluorogenic Mango NASBA
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Peter J. Unrau, Amir Abdolahzadeh, and Elena V. Dolgosheina
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0303 health sciences ,Aptamer ,030302 biochemistry & molecular biology ,Loop-mediated isothermal amplification ,RNA ,Method ,Reproducibility of Results ,Computational biology ,Biology ,Aptamers, Nucleotide ,NASBA ,Sensitivity and Specificity ,03 medical and health sciences ,Nucleic acid ,CRISPR ,Humans ,Fluorometry ,Sensitivity (control systems) ,Molecular Biology ,Nucleic Acid Amplification Techniques ,030304 developmental biology ,Nucleic acid detection ,Fluorescent Dyes - Abstract
There is a pressing need for nucleic acid–based assays that are capable of rapidly and reliably detecting pathogenic organisms. Many of the techniques available for the detection of pathogenic RNA possess one or more limiting factors that make the detection of low-copy RNA challenging. Although RT-PCR is the most commonly used method for detecting pathogen-related RNA, it requires expensive thermocycling equipment and is comparatively slow. Isothermal methods promise procedural simplicity but have traditionally suffered from amplification artifacts that tend to preclude easy identification of target nucleic acids. Recently, the isothermal SHERLOCK system overcame this problem by using CRISPR to distinguish amplified target sequences from artifactual background signal. However, this system comes at the cost of introducing considerable enzymatic complexity and a corresponding increase in total assay time. Therefore, simpler and less expensive strategies are highly desirable. Here, we demonstrate that by nesting NASBA primers and modifying the NASBA inner primers to encode an RNA Mango aptamer sequence we can dramatically increase the sensitivity of NASBA to 1.5 RNA molecules per microliter. As this isothermal nucleic acid detection scheme directly produces a fluorescent reporter, real-time detection is intrinsic to the assay. Nested Mango NASBA is highly specific and, in contrast to existing RNA detection systems, offers a cheap, simple, and specific way to rapidly detect single-molecule amounts of pathogenic RNA.
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- 2019
18. Structural basis for high-affinity fluorophore binding and activation by RNA Mango
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Robert J. Trachman, Shanker Shyam S. Panchapakesan, Adrian R. Ferré-D'Amaré, Natalia A Demeshkina, Peter J. Unrau, Sunny C. Y. Jeng, and Matthew W.L. Lau
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0301 basic medicine ,Fluorophore ,Aptamer ,Ligands ,010402 general chemistry ,01 natural sciences ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Benzothiazoles ,Binding site ,Molecular Biology ,Fluorescent Dyes ,Binding Sites ,Ligand ,RNA ,Cell Biology ,Fluorescence ,0104 chemical sciences ,030104 developmental biology ,Biochemistry ,chemistry ,Quinolines ,Linker ,Macromolecule - Abstract
Genetically encoded fluorescent protein tags revolutionized proteome studies, while the lack of intrinsically fluorescent RNAs has hindered transcriptome exploration. Among several RNA-fluorophore complexes that potentially address this problem, RNA Mango has an exceptionally high affinity for its thiazole orange (TO)-derived fluorophore, TO1-Biotin (Kd ~3 nM), and in complex with related ligands, is one of the most red-shifted fluorescent macromolecular tags known. To elucidate how this small aptamer exhibits such properties, which make it well suited for studying low-copy cellular RNAs, we determined its 1.7 Å resolution co-crystal structure. Unexpectedly, the entire ligand, including TO, biotin, and the linker connecting them, abuts one of the near-planar faces of the three-tiered G-quadruplex. The two heterocycles of TO are held in place by two loop adenines and make a 45° angle with respect to each other. Minimizing this angle would increase quantum yield and further improve this tool for in vivo RNA visualization., Graphical Abstract
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- 2017
19. Live cell imaging of single RNA molecules with fluorogenic Mango II arrays
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David Rueda, Adam D. Cawte, and Peter J. Unrau
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0301 basic medicine ,Fluorophore ,Cell Survival ,Science ,Aptamer ,General Physics and Astronomy ,01 natural sciences ,General Biochemistry, Genetics and Molecular Biology ,Fluorescence ,Article ,chemistry.chemical_compound ,03 medical and health sciences ,0302 clinical medicine ,Single-molecule biophysics ,Live cell imaging ,Molecule ,Humans ,RNA, Messenger ,Biopolymers in vivo ,lcsh:Science ,030304 developmental biology ,0303 health sciences ,Messenger RNA ,Multidisciplinary ,010405 organic chemistry ,Chemistry ,HEK 293 cells ,RNA ,Reproducibility of Results ,General Chemistry ,Aptamers, Nucleotide ,Fibroblasts ,Structured illumination ,Single Molecule Imaging ,Actins ,0104 chemical sciences ,Cell biology ,030104 developmental biology ,HEK293 Cells ,Biophysics ,lcsh:Q ,Extended time ,RNA transport ,Biological fluorescence ,030217 neurology & neurosurgery - Abstract
RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (β-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to bleached fluorophore replacement. This property makes the arrays readily compatible with structured illumination super-resolution microscopy., Fluorogenic RNA aptamers have been used for RNA imaging, but folding and fluorescence stability often limited their use in high resolution applications. Here the authors present an array of stably folding Mango II aptamers for imaging of coding and non-coding RNAs at single-molecule resolution, in both live and fixed cells.
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- 2019
20. Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method
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Quiana R. Ang, Peter J. Unrau, and Iqra M. Yaseen
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RNA, Untranslated ,Fluorophore ,General Chemical Engineering ,Polyacrylamide ,Biotin ,Context (language use) ,Ligands ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Benzothiazoles ,Fluorescent Dyes ,030304 developmental biology ,Ribonucleoprotein ,0303 health sciences ,Mangifera ,General Immunology and Microbiology ,Chemistry ,General Neuroscience ,030302 biochemistry & molecular biology ,RNA ,Fluorescence ,RNA, Bacterial ,Electrophoresis ,Biochemistry ,Quinolines ,Electrophoresis, Polyacrylamide Gel ,Sequence motif - Abstract
Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19-22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.
- Published
- 2019
21. Structure and functional reselection of the Mango-III fluorogenic RNA aptamer
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Robert J. Trachman, Alexis Autour, Sunny C. Y. Jeng, Amir Abdolahzadeh, Alessio Andreoni, Razvan Cojocaru, Ramil Garipov, Elena V. Dolgosheina, Jay R. Knutson, Michael Ryckelynck, Peter J. Unrau, Adrian R. Ferré-D’Amaré, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), and Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)
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0303 health sciences ,[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,Cell Biology ,Aptamers, Nucleotide ,010402 general chemistry ,01 natural sciences ,Article ,0104 chemical sciences ,03 medical and health sciences ,Mutation ,Nucleic Acid Conformation ,Molecular Biology ,Fluorescent Dyes ,030304 developmental biology - Abstract
International audience; Several turn-on RNA aptamers that activate small molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55). Uniquely among related aptamers, Mango-III exhibits biphasic thermal melting, characteristic of molecules with tertiary structure. We report crystal structures of TO1-Biotin complexes of Mango-III, a structure-guided mutant Mango-III(A10U), and a functionally reselected mutant iMango-III. The structures reveal a globular architecture arising from an unprecedented pseudoknot-like connectivity between a G-quadruplex and an embedded non-canonical duplex. The fluorophore is restrained into a planar conformation by the G-quadruplex, a lone, long-range trans-Watson-Crick pair (whose A10U mutation increases quantum yield to 0.66), and a pyrimidine perpendicular to the nucleobase planes of those motifs. The improved iMango-III and Mango-III(A10U) fluoresce ~50% brighter than enhanced green fluorescent protein, making them suitable tags for live cell RNA visualization.
- Published
- 2019
22. Transitions
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Niles Lehman, Peter J. Unrau, and Eric J. Hayden
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education.field_of_study ,biology ,Linear polymer ,Point mutation ,Population ,Ribozyme ,food and beverages ,RNA ,RNA world hypothesis ,Development (topology) ,Evolutionary biology ,biology.protein ,Sequence space (evolution) ,education - Abstract
In this chapter, we discuss the role of RNA in the major transitions that could have occurred during the origin and early evolution of life on the Earth. The ability of RNA to be a catalyst and the existence of ribozymes are important clues to how life developed. We discuss the RNA world hypothesis, and then, with examples of modern-day and laboratory-derived ribozymes, we discuss how RNA can evolve and search sequence space to discover new functions. Because RNA is a linear polymer of consecutive nucleotides, a network of related sequences can exist for any genotype. We describe the evolutionary pressures that exist to move a population of RNAs through this network and consider how similar networks can interact. Both point mutations and recombination contribute to the means by which new genotypes can emerge and evolve. Overall, these considerations depict a series of transitions that retell the history of how chemistry became biology.
- Published
- 2018
23. Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells
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Amir Abdolahzadeh, Adam D. Cawte, Peter J. Unrau, David Rueda, Sunny C. Y. Jeng, Alexis Autour, Angela Galli, Shanker Shyam S. Panchapakesan, Michael Ryckelynck, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), and Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)
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0301 basic medicine ,STRUCTURAL BASIS ,SELECTION ,EXPRESSION ,DYNAMICS ,Science ,Aptamer ,Cells ,[SDV]Life Sciences [q-bio] ,General Physics and Astronomy ,Article ,General Biochemistry, Genetics and Molecular Biology ,Green fluorescent protein ,Cell Line ,03 medical and health sciences ,MIMICS ,Labelling ,Humans ,CRYSTAL-STRUCTURE ,GREEN FLUORESCENT PROTEIN ,lcsh:Science ,SPECIFICITY ,ComputingMilieux_MISCELLANEOUS ,Fluorescent Dyes ,Multidisciplinary ,Science & Technology ,COMPLEX ,Chemistry ,RNA ,General Chemistry ,Aptamers, Nucleotide ,Subcellular localization ,In vitro ,Cell biology ,Multidisciplinary Sciences ,030104 developmental biology ,Microscopy, Fluorescence ,Cell culture ,FLUOROPHORE-BINDING ,Science & Technology - Other Topics ,RNA, Small Untranslated ,lcsh:Q ,Function (biology) - Abstract
Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells., Many aptamer-fluorophore complexes suffer from low quantum yield and low extinction coefficients limiting their usability. Here the authors isolate new Mango aptamers with improved fluorescent properties for both fixed and live-cell imaging by using competitive ligand binding microfluidic selection.
- Published
- 2018
24. Watching U4 snRNA Release During Spliceosome Activation with Mango RNA APTamers
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Aaron A. Hoskins, Peter J. Unrau, and Karli Lipinski
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Spliceosome ,RNA Aptamers ,Chemistry ,Biophysics ,Small nuclear RNA ,Cell biology - Published
- 2021
25. Rapid Clinical Diagnostic Viral Detection with Saliva by a Novel Single Step Nested Mango-NASBA Assay
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Haruki Iino, David Rueda, Artur Kaczmarczyk, Paul Girvan, Amir Abdolahzadeh, Peter J. Unrau, Paul Poudevigne-Durance, George P. Moore, Elena V. Dolgosheina, Tianyi Zhang, and Matthew D. Newton
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Saliva ,Biophysics ,Single step ,Biology ,NASBA ,Virology ,Article - Published
- 2021
26. Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers
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Evan P. Booy, Sean A. McKenna, Hedy H.Y. Chan, Sunny C. Y. Jeng, and Peter J. Unrau
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0301 basic medicine ,Fluorophore ,Aptamer ,Ligands ,010402 general chemistry ,G-quadruplex ,01 natural sciences ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Spinacia oleracea ,Molecular Biology ,Fluorescent Dyes ,Mangifera ,biology ,Temperature ,RNA ,biology.organism_classification ,Ligand (biochemistry) ,Fluorescence ,Small molecule ,0104 chemical sciences ,Spectrometry, Fluorescence ,030104 developmental biology ,chemistry ,Biochemistry ,RNA, Plant ,Spinach - Abstract
The effective tracking and purification of biological RNAs and RNA protein complexes is currently challenging. One promising strategy to simultaneously address both of these problems is to develop high-affinity RNA aptamers against taggable small molecule fluorophores. RNA Mango is a 39-nucleotide, parallel-stranded G-quadruplex RNA aptamer motif that binds with nanomolar affinity to a set of thiazole orange (TO1) derivatives while simultaneously inducing a 103-fold increase in fluorescence. We find that RNA Mango has a large increase in its thermal stability upon the addition of its TO1-Biotin ligand. Consistent with this thermal stabilization, RNA Mango can effectively discriminate TO1-Biotin from a broad range of small molecule fluorophores. In contrast, RNA Spinach, which is known to have a substantially more rigid G-quadruplex structure, was found to bind to this set of fluorophores, often with higher affinity than to its native ligand, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), and did not exhibit thermal stabilization in the presence of the TO1-Biotin fluorophore. Our data suggest that RNA Mango is likely to use a concerted ligand-binding mechanism that allows it to simultaneously bind and recognize its TO1-Biotin ligand, whereas RNA Spinach appears to lack such a mechanism. The high binding affinity and fluorescent efficiency of RNA Mango provides a compelling alternative to RNA Spinach as an RNA reporter system and paves the way for the future development of small fluorophore RNA reporter systems.
- Published
- 2016
27. Fluorophore‐binding <scp>RNA</scp> aptamers and their applications
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Peter J. Unrau and Elena V. Dolgosheina
- Subjects
0301 basic medicine ,Riboswitch ,Genetics ,RNA, Untranslated ,RNA localization ,Trans-acting siRNA ,RNA ,RNA-binding protein ,Computational biology ,Aptamers, Nucleotide ,Biology ,Biochemistry ,Fluorescence ,Epigenesis, Genetic ,03 medical and health sciences ,RNA silencing ,030104 developmental biology ,Transcription (biology) ,Gene expression ,Animals ,Humans ,Transcriptome ,Molecular Biology ,Fluorescent Dyes - Abstract
Why image RNA? Of all the biological molecules, RNA exhibits the most diverse range of functions. Evidence suggests that transcription produces a wide range of noncoding RNAs (ncRNAs), both short (e.g., siRNAs, miRNAs) and long (e.g., telomeric RNAs) that regulate many aspects of gene expression, including the epigenetic processes that underlie cell fate determination, polarization, and morphogenesis. All these functions are realized through the exquisite temporal and spatial control of RNA expression levels and the stability of specific RNAs within well-defined sub-cellular compartments. Given the central importance of RNA in dictating cell behavior via gene-related functions, there is a great demand for RNA imaging methods so as to determine the composition of the cellular ‘transcriptome’ and to acquire a complete spatial-temporal profile of RNA localization. Recent advances in fluorophore-binding RNA aptamers promise to provide exactly this knowledge, which can ultimately advance our understanding of cell function and behavior in conditions of health and disease, and in response to external stimuli. WIREs RNA 2016, 7:843–851. doi: 10.1002/wrna.1383 This article is categorized under: RNA Interactions with Proteins and Other Molecules > Small Molecule–RNA Interactions RNA Methods > RNA Analyses In Vitro and In Silico RNA Methods > RNA Analyses in Cells
- Published
- 2016
28. Structure-Guided Engineering of the Homodimeric Mango-IV Fluorescence Turn-on Aptamer Yields an RNA FRET Pair
- Author
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Robert J. Trachman, Di Wu, Adrian R. Ferré-D'Amaré, Grzegorz Piszczek, Peter J. Unrau, Razvan Cojocaru, Michael Ryckelynck, Architecture et réactivité de l'ARN (ARN), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), and Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0303 health sciences ,Fluorophore ,Base pair ,Aptamer ,030302 biochemistry & molecular biology ,RNA ,Aptamers, Nucleotide ,Article ,G-Quadruplexes ,03 medical and health sciences ,chemistry.chemical_compound ,Förster resonance energy transfer ,chemistry ,Structural Biology ,Fluorescence Resonance Energy Transfer ,Biophysics ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Nucleic acid structure ,Genetic Engineering ,Aptamer Technology ,Molecular Biology ,Systematic evolution of ligands by exponential enrichment ,Fluorescent Dyes ,030304 developmental biology - Abstract
Summary Fluorescent RNA aptamers have been used in cells as biosensor reporters and tags for tracking transcripts. Recently, combined SELEX and microfluidic fluorescence sorting yielded three aptamers that activate fluorescence of TO1-Biotin: Mango-II, Mango-III, and Mango-IV. Of these, Mango-IV was best at imaging RNAs in both fixed and live mammalian cells. To understand how Mango-IV achieves activity in cells, we determined its crystal structure complexed with TO1-Biotin. The structure reveals a domain-swapped homodimer with two independent G-quadruplex fluorophore binding pockets. Structure-based analyses indicate that the Mango-IV core has relaxed fluorophore specificity, and a tendency to reorganize binding pocket residues. These molecular properties may endow it with robustness in the cellular milieu. Based on the domain-swapped structure, heterodimers between Mango-IV and the fluorescent aptamer iSpinach, joined by Watson-Crick base pairing, were constructed. These exhibited FRET between their respective aptamer-activated fluorophores, advancing fluorescent aptamer technology toward multi-color, RNA-based imaging of RNA coexpression and colocalization.
- Published
- 2020
29. Genome-Wide Discovery of Somatic Regulatory Variants in Diffuse Large B-Cell Lymphoma
- Author
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Jordan Davidson, Christopher Rushton, Graham W. Slack, David W. Scott, Daisuke Ennishi, Randy D. Gascoyne, Gregg B. Morin, Joseph M. Connors, Nicole Thomas, Luka Culibrk, Prasath Pararajalingam, Lauren Chong, Ryan D. Morin, Andrew J. Mungall, Steven J. M. Jones, Eric Y. Zhao, Sarah E. Arthur, Christian Steidl, Laura K. Hilton, Aixiang Jiang, Merrill Boyle, Dipankar Sen, Timothy E. Audas, Kevin Bushell, Anja Mottok, Miguel Alcaide, Bruno M. Grande, Barbara Meissner, Selin Jessa, Marco A. Marra, Peter J. Unrau, Pedro Farinha, Sohrab P. Shah, Daniel Lai, Prince Kumar Lat, and Razvan Cojocaru
- Subjects
0301 basic medicine ,Untranslated region ,Science ,General Physics and Astronomy ,FCGR2B ,Biology ,Genome ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,immune system diseases ,Cell Line, Tumor ,hemic and lymphatic diseases ,Genes, Regulator ,medicine ,Humans ,Exome ,lcsh:Science ,Gene ,3' Untranslated Regions ,neoplasms ,Exome sequencing ,Adaptor Proteins, Signal Transducing ,B-Lymphocytes ,Multidisciplinary ,Oncogene ,Genome, Human ,Receptors, IgG ,Genetic Variation ,Nuclear Proteins ,General Chemistry ,Sequence Analysis, DNA ,medicine.disease ,Germinal Center ,3. Good health ,Lymphoma ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,Mutation ,Cancer research ,I-kappa B Proteins ,lcsh:Q ,Lymphoma, Large B-Cell, Diffuse ,Transcriptome ,Diffuse large B-cell lymphoma ,Genome-Wide Association Study - Abstract
Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3′ UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics., The driver mutations for the two main molecular subgroups of diffuse large B-cell lymphoma (DLBCL) are poorly defined. Here, an integrative genomics analysis identifies 3′ UTR NFKBIZ mutations within the activated B-cell DLBCL subgroup and small FCGR2B amplifications in the germinal centre B-cell DLBCL subgroup.
- Published
- 2018
30. Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness
- Author
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Adrian R. Ferré-D'Amaré, Amir Abdolahzadeh, Razvan Cojocaru, Robert J. Trachman, Peter J. Unrau, Alessio Andreoni, Jay R. Knutson, Michael Ryckelynck, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), and Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,Fluorophore ,Aptamer ,[SDV]Life Sciences [q-bio] ,Biotin ,Crystal structure ,010402 general chemistry ,Crystallography, X-Ray ,01 natural sciences ,Biochemistry ,Molecular Docking Simulation ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Benzothiazoles ,Binding site ,ComputingMilieux_MISCELLANEOUS ,Fluorescent Dyes ,Binding Sites ,Chemistry ,RNA ,Aptamers, Nucleotide ,Small molecule ,Fluorescence ,0104 chemical sciences ,030104 developmental biology ,Biophysics ,Quinolines - Abstract
Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates >5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases by 18%, while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analogue studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.
- Published
- 2018
31. FUNCTIONAL CHARACTERIZATION OF NFKBIZ 3′ UTR MUTATIONS IN DIFFUSE LARGE B-CELL LYMPHOMA
- Author
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Daisuke Ennishi, Marco A. Marra, Jordan Davidson, Dipankar Sen, Anja Mottok, C. Steidl, Timothy E. Audas, Christopher Rushton, Kevin Bushell, Randy D. Gascoyne, Aixiang Jiang, David Scott, Razvan Cojocaru, Ryan D. Morin, Gregg B. Morin, Miguel Alcaide, Jean M. Connors, Peter J. Unrau, Sarah E. Arthur, P. Kumar Lat, and Bruno M. Grande
- Subjects
Cancer Research ,Oncology ,Three prime untranslated region ,Chemistry ,medicine ,Hematology ,General Medicine ,medicine.disease ,Diffuse large B-cell lymphoma ,Molecular biology - Published
- 2019
32. Transitioning to DNA genomes in an RNA world
- Author
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Peter J. Unrau and Razvan Cojocaru
- Subjects
0301 basic medicine ,QH301-705.5 ,Science ,Origin of Life ,Biology ,origins of life ,Biochemistry ,Genome ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,ribozyme ,0302 clinical medicine ,RNA polymerase ,None ,reverse transcriptase ,RNA, Catalytic ,Biology (General) ,Polymerase ,Genetics ,General Immunology and Microbiology ,General Neuroscience ,Ribozyme ,RNA ,DNA ,Reverse Transcription ,General Medicine ,Reverse transcriptase ,polymerase ,RNA world hypothesis ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,Medicine ,Insight - Abstract
A highly evolved RNA polymerase ribozyme was found to also be capable of functioning as a reverse transcriptase, an activity that has never been demonstrated before for RNA. This activity is thought to have been crucial for the transition from RNA to DNA genomes during the early history of life on Earth, when it similarly could have arisen as a secondary function of an RNA-dependent RNA polymerase. The reverse transcriptase ribozyme can incorporate all four dNTPs and can generate products containing up to 32 deoxynucleotides. It is likely that this activity could be improved through evolution, ultimately enabling the synthesis of complete DNA genomes. DNA is much more stable compared to RNA and thus provides a larger and more secure repository for genetic information.
- Published
- 2017
33. Ebbie: automated analysis and storage of small RNA cloning data using a dynamic web server.
- Author
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H. Alexander Ebhardt, Kay C. Wiese, and Peter J. Unrau
- Published
- 2006
- Full Text
- View/download PDF
34. RNA complex purification using high-affinity fluorescent RNA aptamer tags
- Author
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Sunny C. Y. Jeng, Peter J. Unrau, and Shanker Shyam S. Panchapakesan
- Subjects
Riboswitch ,0303 health sciences ,Fluorophore ,General Neuroscience ,Aptamer ,030302 biochemistry & molecular biology ,RNA ,Nuclease protection assay ,Plasma protein binding ,Biology ,Fluorescence ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,History and Philosophy of Science ,chemistry ,Biochemistry ,RNA extraction ,030304 developmental biology - Abstract
RNA plays important roles in cellular processes, but RNA-protein complexes are notoriously hard to isolate and study. We compare and contrast existing RNA- and protein-purification strategies with the potential of new RNA-tagging systems such as RNA Spinach and RNA Mango. Each RNA aptamer binds a small fluorophore, resulting in a highly fluorescent complex that is thousands of times brighter than the unbound fluorophore. Provided that the aptamer binding affinity is high enough, derivatized dyes can be used in conjunction with these aptamers to purify RNA complexes while simultaneously using their intrinsic fluorescence to track the complex of interest. The known strengths and weakness of these RNA tagging systems are discussed.
- Published
- 2015
35. Ribonucleoprotein purification and characterization using RNA Mango
- Author
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Xin Chen, Peter J. Unrau, Matthew L. Ferguson, Eric J. Hayden, Aaron A. Hoskins, and Shanker Shyam S. Panchapakesan
- Subjects
0301 basic medicine ,Streptavidin ,RNA, Untranslated ,Aptamer ,Green Fluorescent Proteins ,Biotin ,Method ,Saccharomyces cerevisiae ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,RNA, Small Nuclear ,Native state ,Benzothiazoles ,Molecular Biology ,Polymerase ,Ribonucleoprotein ,biology ,RNA ,Aptamers, Nucleotide ,Molecular biology ,RNA, Bacterial ,030104 developmental biology ,Spectrometry, Fluorescence ,chemistry ,Ribonucleoproteins ,biology.protein ,Quinolines ,Small nuclear RNA - Abstract
The characterization of RNA–protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the S. cerevisiae U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro.
- Published
- 2017
36. Live cell imaging of genomic loci using fluorescent RNA aptamers
- Author
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Peter J. Unrau, David Rueda, Sunny C. Y. Jeng, and Adam D. Cawte
- Subjects
Science & Technology ,02 Physical Sciences ,Cas9 ,Aptamer ,Biophysics ,RNA ,Computational biology ,Biology ,06 Biological Sciences ,Fluorescence ,Molecular biology ,Green fluorescent protein ,Live cell imaging ,CRISPR ,03 Chemical Sciences ,Life Sciences & Biomedicine ,Subgenomic mRNA - Abstract
In recent years, there has been an explosion of SELEX-evolved fluorescent RNA aptamers, such as Spinach and Mango, with enhanced folding, fluorescence and a high affinity for their dyes. These aptamers have a drastically improved fluorescence contrast relative to EGFP and hold great promise for visualising vital cellular processes involving RNA molecules. However, the use of aptamers in live-cell imaging has experienced limited resolution and applicability due to the dynamic nature of RNA. We have recently developed a new method that combines CRISPR/Cas9-based nuclear localization with fluorescent RNA aptamer-based imaging. A major difference with existing approaches is that this system contains an engineered fluorescent RNA aptamer within the sgRNA scaffold in lieu of a fluorescent dCas9-EGFP fusion. This method enables the direct visualisation of genomic loci and their diffusion dynamics within live cells. We anticipate the development of this technology to improve our ability to target specific regions of the genome, as well as to develop multi-colour imaging using different fluorescent sgRNA constructs.
- Published
- 2017
37. Cellular Imaging of Small RNAs using Fluorescent RNA-Mango Aptamers
- Author
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Adam D. Cawte, Sunny C. Y. Jeng, Alexis Autour, David Rueda, Peter J. Unrau, and Michael Ryckelynck
- Subjects
Biochemistry ,010405 organic chemistry ,Chemistry ,Cellular imaging ,Aptamer ,Biophysics ,RNA ,010402 general chemistry ,01 natural sciences ,Fluorescence ,0104 chemical sciences - Published
- 2018
38. High-throughput detection of RNA processing in bacteria
- Author
-
Peter J. Unrau, Robert E. W. Hancock, Geoffrey L. Winsor, Patrick K. Taylor, Shannan J. Ho Sui, Raymond Lo, Luisa S. Chan, Neil Dobson, Raunak Shrestha, Bhavjinder K. Dhillon, Fiona S. L. Brinkman, Cory Spencer, and Erin E. Gill
- Subjects
0301 basic medicine ,lcsh:QH426-470 ,Nucleases ,RNase P ,lcsh:Biotechnology ,030106 microbiology ,Computational biology ,Biology ,Transcriptome ,03 medical and health sciences ,Transcription (biology) ,lcsh:TP248.13-248.65 ,Gene expression ,Genetics ,Transcriptional regulation ,RNA-Seq ,RNA Processing, Post-Transcriptional ,Promoter Regions, Genetic ,030304 developmental biology ,2. Zero hunger ,0303 health sciences ,Sequence Analysis, RNA ,030306 microbiology ,pRNA-Seq ,Chromosome Mapping ,High-Throughput Nucleotide Sequencing ,RNA ,Ribosomal RNA ,Gene regulation ,RNA, Bacterial ,lcsh:Genetics ,RNA processing ,dRNA-Seq ,Pseudomonas aeruginosa ,Transfer RNA ,RNA Cleavage ,Transcription Initiation Site ,Sequence motif ,Transcription ,Genome, Bacterial ,Research Article ,Biotechnology - Abstract
Background Understanding the RNA processing of an organism’s transcriptome is an essential but challenging step in understanding its biology. Here we investigate with unprecedented detail the transcriptome of Pseudomonas aeruginosa PAO1, a medically important and innately multi-drug resistant bacterium. We systematically mapped RNA cleavage and dephosphorylation sites that result in 5′-monophosphate terminated RNA (pRNA) using monophosphate RNA-Seq (pRNA-Seq). Transcriptional start sites (TSS) were also mapped using differential RNA-Seq (dRNA-Seq) and both datasets were compared to conventional RNA-Seq performed in a variety of growth conditions. Results The pRNA-Seq library revealed known tRNA, rRNA and transfer-messenger RNA (tmRNA) processing sites, together with previously uncharacterized RNA cleavage events that were found disproportionately near the 5′ ends of transcripts associated with basic bacterial functions such as oxidative phosphorylation and purine metabolism. The majority (97%) of the processed mRNAs were cleaved at precise codon positions within defined sequence motifs indicative of distinct endonucleolytic activities. The most abundant of these motifs corresponded closely to an E. coli RNase E site previously established in vitro. Using the dRNA-Seq library, we performed an operon analysis and predicted 3159 potential TSS. A correlation analysis uncovered 105 antiparallel pairs of TSS that were separated by 18 bp from each other and were centered on single palindromic TAT(A/T)ATA motifs (likely − 10 promoter elements), suggesting that, consistent with previous in vitro experimentation, these sites can initiate transcription bi-directionally and may thus provide a novel form of transcriptional regulation. TSS and RNA-Seq analysis allowed us to confirm expression of small non-coding RNAs (ncRNAs), many of which are differentially expressed in swarming and biofilm formation conditions. Conclusions This study uses pRNA-Seq, a method that provides a genome-wide survey of RNA processing, to study the bacterium Pseudomonas aeruginosa and discover extensive transcript processing not previously appreciated. We have also gained novel insight into RNA maturation and turnover as well as a potential novel form of transcription regulation. NOTE: All sequence data has been submitted to the NCBI sequence read archive. Accession numbers are as follows: [NCBI sequence read archive: SRX156386, SRX157659, SRX157660, SRX157661, SRX157683 and SRX158075]. The sequence data is viewable using Jbrowse on www.pseudomonas.com. Electronic supplementary material The online version of this article (10.1186/s12864-018-4538-8) contains supplementary material, which is available to authorized users.
- Published
- 2016
39. Electrochemical Sensing Methodology for Antibiogram Assays
- Author
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M. Ash Parameswaran, Shanker Shyam S. Panchapakesan, Shruti Menon, Krishnan Sankaran, Nandimalla Vishnu, Annamalai Senthil Kumar, and Peter J. Unrau
- Subjects
Engineering ,medicine.diagnostic_test ,Renewable Energy, Sustainability and the Environment ,business.industry ,Library science ,Analytical Chemistry (journal) ,Condensed Matter Physics ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Antibiogram ,Materials Chemistry ,Electrochemistry ,medicine ,business - Abstract
aInstitute of Micromachine and Microfabrication Research, School of Engineering Science, Simon Fraser University, Burnaby V5A1S6, Canada bEnvironmental and Analytical Chemistry Division, School of Advanced Sciences, Vellore Institute of Technology University, Vellore 632014, India cInstitute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby V5A1S6, Canada dCentre for Biotechnology, Anna University, Chennai 600025, India
- Published
- 2013
40. Conifers have a unique small RNA silencing signature
- Author
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Ryan D. Morin, S. Cenk Sahinalp, Peter J. Unrau, Jim Mattsson, Elena V. Dolgosheina, Vincent Magrini, Elaine R. Mardis, and Gozde Aksay
- Subjects
Expressed Sequence Tags ,Ribonuclease III ,Genetics ,Small RNA ,RNA, Untranslated ,Molecular Sequence Data ,fungi ,Trans-acting siRNA ,food and beverages ,Plants ,Biology ,Long non-coding RNA ,Evolution, Molecular ,Tracheophyta ,RNA silencing ,RNA, Plant ,RNA interference ,Report ,RasiRNA ,RNA Interference ,Amino Acid Sequence ,Small nucleolar RNA ,Sequence Alignment ,Molecular Biology ,RNA-Directed DNA Methylation - Abstract
Plants produce small RNAs to negatively regulate genes, viral nucleic acids, and repetitive elements at either the transcriptional or post-transcriptional level in a process that is referred to as RNA silencing. While RNA silencing has been extensively studied across the different phyla of the animal kingdom (e.g., mouse, fly, worm), similar studies in the plant kingdom have focused primarily on angiosperms, thus limiting evolutionary studies of RNA silencing in plants. Here we report on an unexpected phylogenetic difference in the size distribution of small RNAs among the vascular plants. By extracting total RNA from freshly growing shoot tissue, we conducted a survey of small RNAs in 24 vascular plant species. We find that conifers, which radiated from the other seed-bearing plants ∼260 million years ago, fail to produce significant amounts of 24-nucleotide (nt) RNAs that are known to guide DNA methylation and heterochromatin formation in angiosperms. Instead, they synthesize a diverse population of small RNAs that are exactly 21-nt long. This finding was confirmed by high-throughput sequencing of the small RNA sequences from a conifer, Pinus contorta. A conifer EST search revealed the presence of a novel Dicer-like (DCL) family, which may be responsible for the observed change in small RNA expression. No evidence for DCL3, an enzyme that matures 24-nt RNAs in angiosperms, was found. We hypothesize that the diverse class of 21-nt RNAs found in conifers may help to maintain organization of their unusually large genomes.
- Published
- 2008
41. RNA complex purification using high-affinity fluorescent RNA aptamer tags
- Author
-
Shanker Shyam S, Panchapakesan, Sunny C Y, Jeng, and Peter J, Unrau
- Subjects
Models, Molecular ,Base Sequence ,Green Fluorescent Proteins ,Nucleic Acid Conformation ,RNA ,Aptamers, Nucleotide ,Binding, Competitive ,Protein Binding ,Protein Structure, Tertiary - Abstract
RNA plays important roles in cellular processes, but RNA-protein complexes are notoriously hard to isolate and study. We compare and contrast existing RNA- and protein-purification strategies with the potential of new RNA-tagging systems such as RNA Spinach and RNA Mango. Each RNA aptamer binds a small fluorophore, resulting in a highly fluorescent complex that is thousands of times brighter than the unbound fluorophore. Provided that the aptamer binding affinity is high enough, derivatized dyes can be used in conjunction with these aptamers to purify RNA complexes while simultaneously using their intrinsic fluorescence to track the complex of interest. The known strengths and weakness of these RNA tagging systems are discussed.
- Published
- 2015
42. Small RNAs derived from tRNAs and rRNAs are highly enriched in exosomes from both old and new world Leishmania providing evidence for conserved exosomal RNA Packaging
- Author
-
Ulrike Lambertz, Mariana Oviedo Ovando, Elton J R Vasconcelos, Peter J. Unrau, Neil E. Reiner, and Peter J. Myler
- Subjects
Small RNA ,RNase P ,Biology ,Exosomes ,Exosome ,Leishmania braziliensis ,03 medical and health sciences ,0302 clinical medicine ,RNA, Transfer ,microRNA ,parasitic diseases ,Genetics ,Animals ,RNA, Messenger ,030304 developmental biology ,Leishmania ,0303 health sciences ,Small non-coding RNA ,Base Sequence ,RNA ,tRNA-derived small RNA ,Ribosomal RNA ,Shuttle RNA ,biology.organism_classification ,Molecular biology ,Cell biology ,MicroRNAs ,030220 oncology & carcinogenesis ,Host cell cytoplasm ,Leishmaniasis, Visceral ,RNA, Small Untranslated ,Biotechnology ,Research Article ,Leishmania donovani - Abstract
Background Leishmania use exosomes to communicate with their mammalian hosts and these secreted vesicles appear to contribute to pathogenesis by delivering protein virulence factors to macrophages. In other eukaryotes, exosomes were found to carry RNA cargo, such as mRNAs and small non-coding RNAs, capable of altering recipient cell phenotype. Whether leishmania exosomes also contain RNAs which they are able to deliver to bystander cells is not known. Here, we show that leishmania exosomes indeed contain RNAs and compare and contrast the RNA content of exosomes released by Leishmania donovani and Leishmania braziliensis. Results We purified RNA from exosomes collected from axenic amastigote culture supernatant and found that when compared with total leishmania RNA, exosomes mainly contained short RNA sequences. Exosomes with intact membranes were capable of protecting their RNA cargo from degradation by RNase. Moreover, exosome RNA cargo was delivered to host cell cytoplasm in vitro. Sequencing of exosomal RNA indicated that the majority of cargo sequences were derived from non-coding RNA species such as rRNA and tRNA. In depth analysis revealed the presence of tRNA-derived small RNAs, a novel RNA type with suspected regulatory functions. Northern blotting confirmed the specific and selective enrichment of tRNA-derived small RNAs in exosomes. We also identified a number of novel transcripts, which appeared to be specifically enriched in exosomes compared to total cell RNA. In addition, we observed the presence of sequences mapping to siRNA-coding regions in L. braziliensis , but not in L. donovani exosomes. Conclusions These results show that leishmania exosomes are selectively and specifically enriched in small RNAs derived almost exclusively from non-coding RNAs. These exosomes are competent to deliver their cargo of novel, potential small regulatory RNAs to macrophages where they may influence parasite-host cell interactions. The remarkably high degree of congruence in exosomal RNA content between L. donovani and L. braziliensis, argues for the presence of a conserved mechanism for exosomal RNA packaging in leishmania. These findings open up a new avenue of research on non-canonical, small RNA pathways in this trypanosomatid, which may elucidate pathogenesis and identify novel therapeutic approaches. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1260-7) contains supplementary material, which is available to authorized users.
- Published
- 2015
- Full Text
- View/download PDF
43. A General RNA-Capping Ribozyme Retains Stereochemistry during Cap Exchange
- Author
-
Hani S. Zaher and Peter J. Unrau
- Subjects
Ribonucleotide ,RNA capping ,Stereochemistry ,Uridine Triphosphate ,RNA Cap Analogs ,Biochemistry ,Pyrophosphate ,Catalysis ,chemistry.chemical_compound ,Adenosine Triphosphate ,Colloid and Surface Chemistry ,Polyphosphates ,RNA, Catalytic ,Nucleotide ,chemistry.chemical_classification ,Base Sequence ,biology ,Chemistry ,GIR1 branching ribozyme ,Ribozyme ,General Chemistry ,Diphosphates ,Kinetics ,Models, Chemical ,Covalent bond ,Isotope Labeling ,biology.protein ,Hairpin ribozyme - Abstract
Numerous natural and artificial ribozymes have been shown to facilitate reactions that invert stereochemistry. Here, we demonstrate that an RNA-capping ribozyme retains stereochemistry at a phosphorus reaction center. The ribozyme synthesizes a broad range of 5'-5' RNA caps by exchanging phosphate groups around the alpha-phosphate found at the 5' terminus of the ribozyme. A ribozyme prepared with an Rp adenosine(5')alpha-thiotetraphosphate cap was found to exchange this cap for an Rp 4-thiouridine(5')alpha-thiotetraphosphate cap when incubated with 4-thiouridine triphosphate. The same Rp capped construct, when incubated with [gamma-(32)P]-ATP, exchanged the unlabeled ATP for a radiolabeled one while maintaining the same stereoconfiguration. In contrast, ribozymes prepared with an Sp cap failed to react even in the presence of thiophilic metal ions such as manganese. The kinetics of capping was also unusual as compared to inverting ribozymes. When the ribozyme was prepared with a triphosphate, capping was found to follow Michaelis-Menten-type kinetics even though the rate of pyrophosphate release was completely independent of nucleotide substrate concentration. Interestingly, the rate of capping and hydrolysis, when summed, was found to be indistinguishable from the rate of pyrophosphate release, indicating that an early rate-limiting step precedes both capping and hydrolysis. Together the retention of stereochemistry and kinetics imply that capping utilizes two inverting chemical steps that are separated by the transient formation of a rate-limiting covalent intermediate. As all protein enzymes that mediate similar capping reactions utilize a covalent intermediate, chemical necessity may have strongly guided the evolution of both protein and RNA-capping catalysts.
- Published
- 2006
44. Recombination During In Vitro Evolution
- Author
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Niles Lehman and Peter J. Unrau
- Subjects
Cell Nucleus ,Recombination, Genetic ,Genetics ,Time Factors ,Models, Genetic ,biology ,Point mutation ,FLP-FRT recombination ,Ribozyme ,Genetic recombination ,Mutation ,biology.protein ,Sequence space (evolution) ,Directed Molecular Evolution ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Systematic evolution of ligands by exponential enrichment ,Recombination ,Selection (genetic algorithm) - Abstract
Recombination, the swapping of large portions of genetic information between and among parental genotypes, can be applied to in vitro evolution experiments on functional nucleic acids. Both homologous and heterologous recombination can be achieved using standard laboratory techniques. In many cases, recombination can allow for the discovery of a ribozyme or DNAzyme phenotype that would not likely be encountered by reliance on point mutations alone. In addition, recombination can often aid in the discovery of global optima in sequence space and/or lessen the number of generations it would take to reach optima. Recombination is most efficiently used in combination with point mutations and applied after the first couple of rounds of selection but before high-fitness genotypes dominate the selection. The "recombination zone" describes that region of sequence space-defined by the residues that will ultimately participate in the function of the winning nucleic acid(s)-where recombination is expected to be the most beneficial in the search for high-fitness genotypes.
- Published
- 2005
45. Isolation of Fast Purine Nucleotide Synthase Ribozymes
- Author
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Matthew W.L. Lau, Kelly E. C. Cadieux, and Peter J. Unrau
- Subjects
Purine ,Stereochemistry ,Population ,Phosphoribosyl Pyrophosphate ,Context (language use) ,Biochemistry ,Catalysis ,Substrate Specificity ,Ligases ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Magnesium ,RNA, Catalytic ,Nucleotide ,education ,Purine Nucleotides ,Protein secondary structure ,Ligase ribozyme ,chemistry.chemical_classification ,education.field_of_study ,biology ,Chemistry ,Ribozyme ,RNA ,General Chemistry ,Thionucleotides ,Guanine Nucleotides ,Kinetics ,biology.protein - Abstract
Here we report the in vitro selection of fast ribozymes capable of promoting the synthesis of a purine nucleotide (6-thioguanosine monophosphate) from tethered 5-phosphoribosyl 1-pyrophosphate (PRPP) and 6-thioguanine ((6S)Gua). The two most proficient purine synthases have apparent efficiencies of 284 and 230 M(-1) min(-1) and are both significantly more efficient than pyrimidine nucleotide synthase ribozymes selected previously by a similar approach. Interestingly, while both ribozymes showed good substrate discrimination, one ribozyme had no detectable affinity for 6-thioguanine while the second had a K(m) of approximately 80 muM, indicating that these ribozymes use considerably different modes of substrate recognition. The purine synthases were isolated after 10 rounds of selection from two high-diversity RNA pools. The first pool contained a long random sequence region. The second pool contained random sequence elements interspersed with the mutagenized helical elements of a previously characterized 4-thiouridine synthase ribozyme. While nearly all of the ribozymes isolated from this biased pool population appeared to have benefited from utilizing one of the progenitor's helical elements, little evidence for more complicated secondary structure preservation was evident. The discovery of purine synthases, in addition to pyrimidine synthases, demonstrates the potential for nucleotide synthesis in an 'RNA World' and provides a context from which to study small molecule RNA catalysis.
- Published
- 2004
46. T7 RNA Polymerase Mediates Fast Promoter-Independent Extension of Unstable Nucleic Acid Complexes
- Author
-
Peter J. Unrau and Hani S. Zaher
- Subjects
Base pair ,RNA-dependent RNA polymerase ,Biology ,Biochemistry ,Nucleic acid secondary structure ,Viral Proteins ,chemistry.chemical_compound ,Biopolymers ,Transcription (biology) ,Nucleic Acids ,RNA polymerase ,medicine ,RNA polymerase I ,T7 RNA polymerase ,Phosphorylation ,Promoter Regions, Genetic ,Base Pairing ,Polymerase ,DNA Primers ,DNA-Directed RNA Polymerases ,Templates, Genetic ,Kinetics ,chemistry ,biology.protein ,medicine.drug - Abstract
T7 RNA polymerase is a processive, DNA-dependent RNA polymerase that has a high specificity for its 17 base pair (bp) promoter. In addition to normal transcription, the enzyme can produce anomalous transcripts in the absence of a promoter. We report here the systematic characterization of the transient aspects of this promoter-independent process. Oligonucleotides that are able to form transient unimolecular loop structures closed by as little as one Watson-Crick base pair between the 3' terminal residue and an internal nucleotide proved to be viable substrates. A single nucleotide triphosphate assay system found that incorporation was encoded by the nucleotide 5' to the predicted transient base pair. When this coding nucleotide was identical to the internal nucleotide participating in the transient base pair, multiple nucleotide incorporations were observed and could only be explained by a continuous shifting and resetting of the transient base-pairing required for extension. This intermittent extension process can be quite efficient. Short DNA or RNA substrates were good substrates for the enzyme (affinities ranged from 2 to 43 microM) and were extended rapidly with apparent catalytic rates of up to 240 min(-1) being observed, only 2-fold slower than the rate of transcriptional initiation. Our data suggest a possible mechanism for this promoter-independent extension activity and may add to the understanding of viral RNA replicative strategies.
- Published
- 2004
47. An oxocarbenium-ion intermediate of a ribozyme reaction indicated by kinetic isotope effects
- Author
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Peter J. Unrau and David P. Bartel
- Subjects
chemistry.chemical_classification ,Binding Sites ,Multidisciplinary ,Base Sequence ,biology ,Stereochemistry ,GIR1 branching ribozyme ,Molecular Sequence Data ,Reactive intermediate ,Ribozyme ,Oxocarbenium ,Leaving group ,Active site ,Biological Sciences ,Photochemistry ,Catalysis ,Kinetics ,chemistry ,Phosphodiester bond ,biology.protein ,Pyrimidine Nucleotides ,RNA, Catalytic ,Nucleotide ,Glycosides - Abstract
Many of the enzymes that catalyze reactions at nucleotide glycosidic linkages proceed through either a reactive oxocarbenium-ion intermediate or a transition state with considerable oxocarbenium character. To investigate how an RNA active site deals with the catalytic challenge of nucleotide synthesis, we probed the transition state of a ribozyme able to promote the formation of a pyrimidine nucleotide. Primary and secondary kinetic isotope effects indicate that this ribozyme stabilizes a highly dissociative reaction with considerable sp 2 hybridization and negligible bond order between the departing pyrophosphate leaving group and the anomeric carbon. The small primary 13 C isotope effect of 1.002 ± 0.003 indicates that the reaction is likely to be less concerted than that observed for protein nucleotide synthesis enzymes, which typically have primary 13 C isotope effects of 1.02–1.03. The dissociative nature of the ribozyme reaction most resembles the reaction of some hydrolytic enzymes, such as uracil DNA glycosylase, which uses the negative charges found in the phosphodiester backbone of its DNA substrate to transiently stabilize an oxocarbenium ion during hydrolysis. The detectable hydrolysis observed in the ribozyme reaction indicates that shielding of this reactive intermediate from water is a significant challenge for RNA, which protein enzymes that synthesize nucleotides have managed to overcome during evolution, apparently by the utilization of more concerted chemistry.
- Published
- 2003
48. Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme
- Author
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Kelly E. Chapple, Peter J. Unrau, and David P. Bartel
- Subjects
Sequence analysis ,Molecular Sequence Data ,Computational biology ,Article ,Substrate Specificity ,Sequence Homology, Nucleic Acid ,RNA, Catalytic ,Molecular Biology ,Protein secondary structure ,DNA Primers ,Sequence Deletion ,Genetics ,Binding Sites ,Base Sequence ,biology ,Nucleotides ,GIR1 branching ribozyme ,Ribozyme ,RNA Ligase (ATP) ,Kinetics ,Amino Acid Substitution ,biology.protein ,Nucleic acid ,Nucleic Acid Conformation ,Hairpin ribozyme ,Pseudoknot ,VS ribozyme - Abstract
We previously isolated from random sequences ribozymes able to form a glycosidic linkage between a ribose sugar and 4-thiouracil in a reaction that mimics protein-catalyzed nucleotide synthesis. Here we report on two serial in vitro selection experiments that defined the core motif of one of the nucleotide synthase ribozymes and provided improved versions of this ribozyme. The first selection experiment started from a degenerate sequence pool based on the previously isolated sequence and used a selection-amplification protocol that allowed the sequence requirements at the 3′ terminus of the ribozyme to be interrogated. Comparing the active sequences identified in this experiment revealed the complicated secondary structure of the nucleotide synthase ribozyme. A second selection was then performed to remove nonessential sequence from the ribozyme. This selection started with a pool with variation introduced in both the sequence and the length of the nonconserved loops and joining regions. This pool was generated using a partial reblocking/deblocking strategy on a DNA synthesizer, allowing the combinatorial synthesis of both point deletions and point substitutions. The consensus ribozyme motif that emerged was an ~71 nt pseudoknot structure with five stems and two important joining segments. Comparative sequence analysis and a cross-linking experiment point to the probable location of nucleotide synthesis. The prototype isolate from the second selection was nearly 35 times more efficient than the initial isolate and at least 108 times more efficient than an upper limit of an as-yet undetectable uncatalyzed reaction, supporting the idea that RNA-catalyzed nucleotide synthesis might have been important in an RNA world.
- Published
- 2003
49. Purification of Histidine-Tagged T4 RNA Ligase from E. coli
- Author
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Peter J. Unrau and Qing S. Wang
- Subjects
chemistry.chemical_classification ,Expression vector ,RNase P ,Biology ,medicine.disease_cause ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,Sepharose ,Enzyme ,chemistry ,Gene expression ,medicine ,Escherichia coli ,Histidine ,Biotechnology ,RNA ligase - Abstract
Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1–3 mg pure protein/g cell pellet. A simple purification procedure ensures that the enzyme is de-adenylated to levels comparable to those found for many commercial preparations. The purified protein has very low levels of RNase contamination and functioned normally in a variety of activity assays.
- Published
- 2002
50. In Vitro Binding of 6S RNA Mango to RNA Polymerase by Two Photon Fluorescence Cross Correlation Spectroscopy
- Author
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Eric J. Hayden, Peter J. Unrau, S. Shyam Sundar Panchapakesan, and Matthew L. Ferguson
- Subjects
Aptamer ,Biophysics ,Chemical biology ,RNA ,Biology ,Binding constant ,Molecular biology ,In vitro ,Green fluorescent protein ,chemistry.chemical_compound ,Biochemistry ,chemistry ,In vivo ,RNA polymerase - Abstract
The assembly of RNA and protein complexes is a fundamental process for life but has been difficult to study both in vitro and in vivo. Recently a novel aptamer based method to fluorescently label RNA molecules has been reported(Dolgosheina et al. 2014). Here we report the successful utilization of the RNA Mango + thiazole orange derivative to measure the in vitro binding of 6S RNA to RNA Polymerase from E. coli. By two photon Fluorescence Cross Correlation Spectroscopy, we are able to independently measure the molecular brightness, diffusion coefficient and concentration of 6S RNA Mango, RNA Polymerase GFP and complex as we increase RNA Polymerase concentration giving us an estimate for the equilibrium binding constant of the complex in vitro. This experiment demonstrates the utility of RNA Mango for both in vitro and in vivo single molecule experiments.Dolgosheina, Elena V., Sunny C. Y. Jeng, Shanker Shyam S. Panchapakesan, Razvan Cojocaru, Patrick S. K. Chen, Peter D. Wilson, Nancy Hawkins, Paul A. Wiggins, and Peter J. Unrau. 2014. “RNA Mango Aptamer-Fluorophore: A Bright, High-Affinity Complex for RNA Labeling and Tracking.” ACS Chemical Biology 9 (10): 2412-20.
- Published
- 2017
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