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Development, Characterization, and Application of RNA Catalysts for in Situ Labeling of Target RNA Molecules

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Development, Characterization, and Application of RNA Catalysts for in Situ Labeling of Target RNA Molecules Development, characterization, and application of RNA catalysts for in situ labeling of target RNA molecules Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program IMPRS Molecular Biology of the Georg-August University School of Science (GAUSS) submitted by Mohammad Ghaem Maghami from Tehran, Iran Göttingen, June 2020 Thesis Committee Prof. Dr. Claudia Höbartner, Organic and Biomolecular Chemistry, Institute for Organic Chemistry, University of Würzburg. Prof. Dr. Marina Rodnina, Biophysical Chemistry, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Stefan Jakobs, Mitochondrial Structure and Dynamics, Max Planck Institute for Biophysical chemistry, Göttingen Members of the Examination Board Referee: Prof. Dr. Claudia Höbartner, Organic and Biomolecular Chemistry, Institute for Organic Chemistry, University of Würzburg. 2nd Referee: Prof. Dr. Marina Rodnina, Biophysical Chemistry, Max Planck Institute for Biophysical Chemistry, Göttingen. Further members of the Examination Board Prof. Dr. Stefan Jakobs, Mitochondrial Sructure and Dynamics, Max Planck Institute for Biophysical Chemistry, Göttingen. Prof. Dr. Patrick Cramer, Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen. Prof. Dr. Markus Bohnsack, Molecular Biology, University Medical Center Göttingen. Prof. Dr. Kai Tittmann, Molecular Enzymology, Göttingen Center for Molecular Biology (GZMB). Date of oral examination: 06.07.2020 i Herewith I declare that I prepared the doctoral thesis "Development, characterization, and application of RNA catalysts for in situ labeling of target RNA molecules" on my own and with no other sources and aids than quoted. This thesis has not been submitted elsewhere for any academic award or qualification. Mohammad Ghaem Maghami. Göttingen, 27.05.2020 ii Table of content Summary ........................................................................................................................................... 1 1 Introduction .............................................................................................................................. 3 1.1 Non-catalytic role of RNA ................................................................................................. 3 1.2 RNA as catalyst .................................................................................................................. 4 1.3 Strategies for development of artificial RNA catalysts ...................................................... 5 1.3.1 Selection using transition state analogs .......................................................................... 6 1.3.2 Selection based on tagging of the active species ............................................................ 7 1.3.2.1 Addition of oligonucleotide tags .................................................................................... 7 1.3.2.2 Tagging by circularization.............................................................................................. 8 1.3.2.3 Affinity tagging .............................................................................................................. 8 1.3.2.3.1 Thiol-reactive capture of the active species ............................................................. 10 1.3.2.3.2 Biotin as a capture tag .............................................................................................. 11 1.3.2.4 Selection using other tags ............................................................................................. 15 1.3.3 Selection based on in vitro compartmentalization........................................................ 16 1.4 RNA imaging methods ..................................................................................................... 17 1.4.1 Non-covalent labeling methods .................................................................................... 18 1.4.1.1 Hybridization based methods ....................................................................................... 18 1.4.1.1.1 FISH ......................................................................................................................... 18 1.4.1.1.2 Molecular beacon ..................................................................................................... 18 1.4.1.2 RNA binding protein-based methods ........................................................................... 19 1.4.1.2.1 MS2-tagging ............................................................................................................. 19 1.4.1.2.2 Pumilio domain ........................................................................................................ 20 1.4.1.2.3 CRISPR-Cas based systems ..................................................................................... 21 1.4.1.3 Fluorogenic aptamers ................................................................................................... 23 1.4.1.3.1 GFP fluorophore analog binding aptamers .............................................................. 24 1.4.1.3.2 Thiazole-orange binding aptamers ........................................................................... 25 1.4.1.3.3 Silicon rhodamine binding aptamer ......................................................................... 25 1.4.1.3.4 DNB aptamers .......................................................................................................... 26 1.4.1.3.5 BHQ aptamers .......................................................................................................... 26 1.4.1.3.6 SRB-2 aptamer ......................................................................................................... 26 1.4.1.3.7 o-Coral ...................................................................................................................... 27 1.4.1.3.8 Riboglow .................................................................................................................. 27 1.4.2 Covalent methods ......................................................................................................... 28 1.4.2.1 Polymerase mediated incorporation of labeled nucleotides ......................................... 28 1.4.2.2 RNA modifying enzymes ............................................................................................. 29 1.4.2.2.1 RNA methyltransferases .......................................................................................... 29 iii 1.4.2.2.2 Other tRNA-modifying enzymes ............................................................................. 31 1.4.2.3 Catalytic nucleic acid-based RNA labeling .................................................................. 32 1.4.2.3.1 Ribozyme based methods ......................................................................................... 32 1.4.2.4 Deoxyribozyme based RNA labeling methods ............................................................ 35 2 Aim of the study...................................................................................................................... 37 3 Results ..................................................................................................................................... 39 3.1 General design of the selection strategy ........................................................................... 39 3.1.1 The design of the selection pool ................................................................................... 39 3.1.2 Construction of the selection pool ................................................................................ 39 3.1.3 Overview of the selection process ................................................................................ 40 3.2 Selection of the nucleotidyltransferase ribozymes ........................................................... 44 3.2.1 Selection process .......................................................................................................... 44 3.2.2 Activity assay on the enriched pool ............................................................................. 45 3.2.2.1 Cis-activity assay .......................................................................................................... 45 3.2.2.2 Trans-activity assay of the pool.................................................................................... 46 3.2.3 Cloning of the enriched pool and activity assay of the individual clones .................... 47 3.2.4 Sanger sequencing results ............................................................................................ 48 3.2.5 Kinetic comparison of the evolved species .................................................................. 49 3.2.6 Secondary structure prediction of the evolved variants ............................................... 50 3.2.7 Characterization of the reaction product ...................................................................... 51 3.2.7.1 Probing of the modification-site ................................................................................... 51 3.2.7.2 ESI-Mass spectrometry of the reaction product ........................................................... 52 3.2.8 Characterization of the FH14 and FH31 ribozymes ..................................................... 53 3.2.8.1 Target sequence scope of the evolved variants ............................................................ 53 3.2.8.1.1 Substrate sequence specificity of FH14 and FH31..................................................
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