In vitro Evolution of a Processive Clamping RNA Polymerase Ribozyme with Promoter Recognition by Razvan Cojocaru BSc, Simon Fraser University, 2014 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Molecular Biology and Biochemistry Faculty of Science © Razvan Cojocaru 2021 SIMON FRASER UNIVERSITY Summer 2021 Copyright in this work is held by the author. Please ensure that any reproduction or re-use is done in accordance with the relevant national copyright legislation. Declaration of Committee Name: Razvan Cojocaru Degree: Doctor of Philosophy Title: In vitro Evolution of a Processive Clamping RNA Polymerase Ribozyme with Promoter Recognition Committee: Chair: Lisa Craig Professor, Molecular Biology and Biochemistry Peter Unrau Supervisor Professor, Molecular Biology and Biochemistry Dipankar Sen Committee Member Professor, Molecular Biology and Biochemistry Michel Leroux Committee Member Professor, Molecular Biology and Biochemistry Mani Larijani Internal Examiner Associate Professor, Molecular Biology and Biochemistry Gerald Joyce External Examiner Professor, Jack H. Skirball Center for Chemical Biology and Proteomics Salk Institute for Biological Studies Date Defended/Approved: August 12, 2021 ii Abstract The RNA World hypothesis proposes that the early evolution of life began with RNAs that can serve both as carriers of genetic information and as catalysts. Later in evolution, these functions were gradually replaced by DNA and enzymatic proteins in cellular biology. I start by reviewing the naturally occurring catalytic RNAs, ribozymes, as they play many important roles in biology today. These ribozymes are central to protein synthesis and the regulation of gene expression, creating a landscape that strongly supports an early RNA World. Ribozymes have also been produced in the laboratory using artificial rather than natural selection. As phosphoryl transfer reactions are central to the energy balance of all organisms in modern biology, I explore artificially selected kinase, glycosidic bond forming, capping, ligase, and polymerase ribozymes, highlighting the importance of phosphoryl transfer reactions from nucleotide and nucleoside metabolism to the assembly and replication of RNA molecules in an RNA World setting. RNA replicases capable of general and self-replication are thought to have been essential early in evolution. However, how such sophisticated polymerases evolved to enable processive gene expression remains largely unexplored. I performed a complex selective strategy that screened ~1013 pool variants to isolate a three domain holopolymerase ribozyme, containing a class I ligase catalytic core, an NTP positioning accessory domain, and a processivity clamping domain. This ribozyme uses a sigma factor–like specificity primer to first recognize an RNA promoter sequence, and then, in a second step, rearrange to a processive clamped elongation form. When correctly assembled, the clamped complex results in more than one order of magnitude increase in extension, synthesizing duplexes of 50-107 base pairs in size. The polymerase can also synthesize part of its own specificity primer, programming itself to polymerize from certain RNA promoters and not others, demonstrating how RNA polymerase ribozymes could have preferentially replicated their own genomes and associated genes, while avoiding replicative parasites in a primordial RNA World. The clamp-like mechanism of my selected polymerase could eventually enable strand displacement and improve fidelity, both being critical requirements for replication in the early evolution of life. Keywords: RNA World; in vitro evolution; ribozyme; RNA polymerase; promoter recognition; processivity iii Acknowledgements First and foremost, I would like to thank my senior supervisor, Dr. Peter Unrau, for taking me under his supervision and providing me with the support and guidance needed to become the scientist I am today. Thank you for all the opportunities you have given me over the years and the many thought-provoking conversations we have shared about science and career. Thank you to my committee members, Dr. Dipankar Sen and Dr. Michel Leroux, for their encouragement and for taking the time and interest to be involved in my projects, providing invaluable feedback and perspectives. I would also like to thank my internal examiner, Dr. Mani Larijani, and my external examiner, Dr. Gerald Joyce, for taking the time to read my thesis and to participate in my defence. I would like to thank all the collaborators I have had the pleasure of working with over these past years. Thank you to Dr. Michael Ryckelynck and his lab for their hospitality. Thank you to the research team at Lumex Instruments, as well as to Dr. Ryan Morin and Dr. Adrian Ferré-D'Amaré and their laboratories, for the fruitful collaborations. Thank you to the MBB office and teaching staff for all their help and support. I would also like to thank fellow graduate students and friends I have journeyed with throughout the years in Dr. Unrau’s lab: Dr. Sunny Jeng, Dr. Shyam Panchapakesan, Amir Abdolahzadeh, Iqra Yaseen, Florian Weissenboeck, Kristen Kong, Christopher Bonar, Dr. Lena Dolgosheina, Dr. Mariana Oviedo, Lyssa Martin and all the undergraduate and high school students passing through, for moral support and meaningful discussions. I would like to thank my family and friends for their continuous love and support, as well as for the many philosophical conversations shared over fermented beverages. A special thank you to my parents, Gabriela and Aurel Cojocaru, and sister, Anca, for their infinite supply of love, patience and encouragement throughout my life. Without you I would not be where I am today. Lastly, I would like to thank my partner, Caelie Stewart, for always being by my side and all the joy, love and support she has provided me throughout this journey. Thank you for being ready at a moment’s notice to celebrate the smallest successes and lift me up from the inevitable falls of graduate studies. iv Preface: Short Summary of Contributions not Included in the Thesis Over the course of my PhD, my research has spanned four RNA based projects of significance: 1. Exploring the origin of life by using in vitro evolution to develop a processive RNA polymerase ribozyme; 2. Validating a microchip real-time PCR technology for detection of SARS-CoV-2 in clinical samples, that uses 10-fold less reagents than the current CDC approved PCR detection tests; 3. Development and characterization of RNA Mango fluorogenic aptamers; And 4. Structural investigation of the NFKBIZ gene 3' untranslated region, mutations in which are linked to diffuse large B- cell lymphoma. However, since the majority of my time and focus has been on the origin of life, I, together with my supervisors, have decided to focus the thesis entirely on this subject matter. Nevertheless, I will briefly summarize all the contributions not included in this thesis. Validation of a microchip RT-PCR technology for SARS-CoV-2 detection 1. Cojocaru, R., Yaseen, I., Unrau, P.J., Lowe, C.F., Ritchie, G., Romney, M.G., Sin, D.D., Gill, S., Slyadnev, M., 2021. Microchip RT-PCR Detection of Nasopharyngeal SARS-CoV-2 Samples. J Mol Diagn 23, 683–690. Fast, accurate, and reliable diagnostic tests have been 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 reported 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 (CDC) approved real-time RT-PCR assays with comparable sensitivity, v showing 100% positive and negative predictive agreement of clinical samples. I contributed to this project by performing half the experiments and manuscript writing. Development and characterization of RNA Mango fluorogenic aptamers 2. Trachman, R.J., Abdolahzadeh, A., Andreoni, A., Cojocaru, R., Knutson, J.R., Ryckelynck, M., Unrau, P.J., Ferré-D’Amaré, A.R., 2018. Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness. Biochemistry 57, 3544–3548. Several RNA aptamers that bind small molecules and enhance their fluorescence have been selected in vitro and used successfully to tag and track RNAs in vivo. Recently, combined SELEX and microfluidic fluorescence sorting yielded three aptamers that activate fluorescence of TO1-Biotin: Mango-II, Mango-III, and Mango-IV (Autour et al., 2018). Of the three, Mango-II, binds TO1-Biotin with the highest affinity, ∼1 nM, and enhances its fluorescence by >1500-fold. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin (Em: 535 nm) and a more significantly red shifted TO3-Biotin (Em: 658