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SFU Thesis Template Files Towards a Prebiotically Plausible Mechanism for the Emergence of RNA, Ribozymes and the RNA World by Lyssa Lynn Martin B.Sc., Thompson Rivers University, 2011 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Department of Molecular Biology and Biochemistry Faculty of Science Lyssa Lynn Martin 2015 SIMON FRASER UNIVERSITY Summer 2015 Approval Name: Lyssa Lynn Martin Degree: Master of Science Title: Towards a Prebiotically Plausible Mechanism for the Emergence of RNA, Ribozymes and the RNA World Examining Committee: Chair: Dr. Fredric Pio Associate Professor Peter Unrau Senior Supervisor Professor Dipankar Sen Supervisor Professor Niles Lehman Supervisor Professor Portland State University Department of Chemistry Nancy Forde Internal Examiner Associate Professor Department of Physics Date Defended/Approved: August 25, 2015 ii Abstract Available evidence, both in vitro and in vivo, attests to the descent of life from the RNA World; however, the prebiotic genesis of such RNA life remains ambiguous. How did The RNA World emerge from the abiotic chemistry on the Archean Earth? Montmorillonite clays have been shown to catalyze the polymerization of activated nucleotides (eg. adenosine 5′ phosphorimidazolide) into RNA, but polymerization has not previously been demonstrated for a prebiotically plausible nucleotide such as cyclic 2′, 3′-adenosine monophosphate (A>p). I reacted A>p in the presence of montmorillonite clay and could detect RNA polymers up to 5 nucleotides in length using a combination of HPLC, enzymatic labeling and mass spectrometry. This chemistry was found to be sensitive to pH and temperature. Reactions at pH 6 were found to produce more polymerization products than reactions at pH 7 or 8. Similarly A>p was found to be very stable at pH 6 with a hydrolysis rate at 25ºC -9 -1 -10 -1 of 6.2 x 10 sec (t1/2 = 3.5 years) and at 4ºC of 4.4 x 10 sec (t1/2 = 50 years). Also discussed are the requirements for the transition from abiotic chemistry to life. Important considerations for the emergence of replicating RNA networks from randomly synthesized RNA are outlined. Finally, the progress towards recreating the RNA world in the laboratory is summarized. Keywords: RNA, RNA World Hypothesis; abiogenesis; origin of life; nucleotide chemistry; montmorillonite clay iii Dedication For Sean, the light of my life and my partner for all adventure. For my parents, who gave me this life and whose love, dedication and wisdom I am grateful for beyond measure. For my family and friends, without whom I would surely have lost my sanity. And for my mentors past and present, who make me better by challenging me, even though I rarely appreciate it at the time. iv Acknowledgements Thank you Sean, Mom, Dad and family for your unwavering love and support, I would be lost without you. I would like to thank Dr. Peter Unrau and the members of the Unrau lab: Marianna, Lena, Neil, Shyam, Sunny, Razvan, Matt, Tim, Irene, Vy, Stephanie, Bashe, Ibrahim, and Amir for their support and comradery. Thank you to my committee members Dr. Dipankar Sen and Dr. Niles Lehman, and also to Dr. Clint Spigel for their help feedback and suggestions. I would also like to thank the American Colloid Company for their gift of clay samples and Dr. James Ferris for advice in preparing those samples. Thank you to all of the MBB grad caucus members: Kaylee, Even, Vil, Laura, Jesper, Julie R, Dipa, Sharron, Aarti, Mike, Bruno, Thea, Julie S, Saeideh, Zoreh, Razvan and Navi for all the good times and for providing a safe haven to hide from experiments that just won′t work. A very special thank you to Evan and Katie Quon for providing power and wi-fi the weekend before this thesis was due, after half the city lost electricity for days. Thank you to the Sen, Cornell, Beh and Hawkins labs; the staff in the MBB office and anyone else I may have missed. v Table of Contents Approval .......................................................................................................................... ii Abstract .......................................................................................................................... iii Dedication ...................................................................................................................... iv Acknowledgements ......................................................................................................... v Table of Contents ........................................................................................................... vi List of Tables ................................................................................................................. viii List of Figures................................................................................................................. ix List of Acronyms ............................................................................................................. xi Chapter 1. The Origin of Life ..................................................................................... 1 1.1. The RNA World Hypothesis: a biochemical theory of our origins ............................ 1 1.2. Arguments against alternative hypotheses ............................................................. 4 1.3. Before RNA: the prebiotically plausible synthesis of nucleotides ............................ 7 Chapter 2. The Origin of RNA polymers ................................................................. 10 2.1. From nucleotides to RNA: the role of mineral surfaces ......................................... 10 2.2. Nomenclature of RNA oligomers .......................................................................... 12 2.3. Selection of Clay ................................................................................................... 14 2.3.1. Introduction .............................................................................................. 14 2.3.2. Methods .................................................................................................. 14 Description of clays ................................................................................................ 14 Optical properties of clays ...................................................................................... 15 Nucleic acid binding capacity ................................................................................. 15 Polymerization of ImpA .......................................................................................... 16 2.3.3. Results .................................................................................................... 18 Optical properties of clay ....................................................................................... 18 Nucleic acid binding capacity ................................................................................. 18 Polymerization of ImpA .......................................................................................... 19 2.3.4. Selection of clay: S/C Yellow ................................................................... 20 2.4. Determination of A>p stability ............................................................................... 21 2.4.1. Introduction .............................................................................................. 21 2.4.2. Methods .................................................................................................. 22 Determination of A>p pH stability .......................................................................... 22 Hydrolysis of A>p at pH 6 ...................................................................................... 22 2.4.3. Results .................................................................................................... 23 Determination of A>p pH stability .......................................................................... 23 Hydrolysis of A>p at pH 6 ...................................................................................... 25 2.5. The pH dependence of A>p polymerization on clay .............................................. 27 2.5.1. Introduction .............................................................................................. 27 2.5.2. Methods .................................................................................................. 29 2.5.3. Results .................................................................................................... 29 2.5.4. Selection of pH 6 ..................................................................................... 30 2.6. Elution of RNA from Clay ...................................................................................... 31 2.6.1. Introduction .............................................................................................. 31 2.6.2. Methods .................................................................................................. 31 vi Binding of RNA oligomers to clay .......................................................................... 31 Elution of RNA oligomers from clay ....................................................................... 32 Comparison to Joshi et al. 2009 method of elution ................................................ 32 2.6.3. Results .................................................................................................... 33 Binding of RNA oligomers to clay .......................................................................... 33 Elution of RNA oligomers from clay
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