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Open Thesisetd.Pdf The Pennsylvania State University The Graduate School Eberly College of Science ENHANCING RNA CATALYSIS THROUGH COMPARTMENTALIZATION A Thesis in Chemistry by Rosalynn Molden Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2009 The thesis of Rosalynn Molden was reviewed and approved* by the following: Christine Keating Associate Professor of Chemistry Thesis Co-Adviser Philip Bevilacqua Professor of Chemistry Thesis Co-Adviser Scott Showalter Assistant Professor of Chemistry Paul Weiss Distinguished Professor of Chemistry and Physics Barbara Garrison Shapiro Professor of Chemistry Head of the Department of Chemistry *Signatures are on file in the Graduate School iii ABSTRACT Experimental models of cellular conditions are useful because they can be used to study cell processes in a controlled environment and because they can be used to model prebiotic conditions. The influence of macromolecular crowding and compartmentalization on the cleavage rates of bimolecular minimal hammerhead ribozymes was investigated using aqueous two-phase systems (ATPS) as a model of the cell cytoplasm. The ATPS consisted of poly(ethyleneglycol) (PEG), dextran, 10 mM MgCl2 and buffer. The PEG-rich and dextran-rich phases of the ATPS segregate to create chemically distinct environments which cause differences in the local concentrations of RNA and provide a macromolecularly crowded environment similar to the cell cytoplasm. It was found that RNA concentrates to the dextran-rich phase of a PEG/dextran ATPS in a length-dependent manner and that long RNA sequences localize almost completely to the dextran-rich phase. It was also found that the local concentration of ribozyme in the dextran-rich phase of an ATPS could be controlled by changing the volume ratio of dextran-rich phase to PEG-rich phase. For most of the hammerhead ribozymes studied, increasing the local concentration of ribozyme also increased the observed rate of cleavage. There was a maximum of a 20-fold enhancement in the observed cleavage rate for 2 nM hammerhead ribozyme in a 250 µL ATPS with a 1:100 dextran to PEG-rich phase ratio compared to 2 nM hammerhead ribozyme in just dextran-rich phase. This study shows that compartmentalization can enhance RNA function, which may be important in RNA therapeutics, study of the RNA world, and further developing ATPS as a primitive model of the cell. iv TABLE OF CONTENTS LIST OF FIGURES AND SCHEMES........................................................................................ v LIST OF TABLES........................................................................................................................ vii ACKNOWLEDGEMENTS......................................................................................................... viii INTRODUCTION........................................................................................................................ 1 RNA compartmentalization ................................................................................................. 1 Aqueous two-phase systems ................................................................................................ 2 RNA partitioning.................................................................................................................. 3 Hammerhead ribozyme ........................................................................................................ 3 Enhanced RNA catalysis through partitioning in ATPS.................................................... 5 MATERIALS AND METHODS ................................................................................................ 6 Abbreviations........................................................................................................................ 6 Materials................................................................................................................................ 6 DNA and RNA sequences ................................................................................................... 6 Aqueous two-phase systems ................................................................................................ 8 RNA preparation .................................................................................................................. 8 RNA partitioning.................................................................................................................. 9 Determining the length dependence of the partitioning coefficient.................................. 11 Hammerhead ribozyme kinetics .......................................................................................... 11 RNA concentration predictions in ATPS............................................................................ 14 RESULTS… ................................................................................................................................. 16 Nucleic acid partitioning trends in ATPS ........................................................................... 16 Length-dependence of RNA partitioning............................................................................ 19 Characterization of ELHH kinetics........................................................................................ 22 Enhanced catalysis of ELHH in ATPS.................................................................................. 25 Enhanced catalysis of other ribozymes............................................................................... 28 DISCUSSION............................................................................................................................... 31 Development of techniques for determining RNA partitioning coefficients in ATPS .... 31 RNA partitioning trends....................................................................................................... 32 Influence of macromolecular crowding on hammerhead ribozyme kinetics.................... 33 Enhanced catalysis ............................................................................................................... 35 Compartmentalization .......................................................................................................... 37 Implications for the RNA world.......................................................................................... 39 FUTURE DIRECTIONS ............................................................................................................. 41 CONCLUSIONS .......................................................................................................................... 43 REFERENCES ............................................................................................................................. 44 v LIST OF FIGURES AND SCHEMES Figure 1: Structures of PEG and dextran. ................................................................................... 3 Figure 2: Hammerhead ribozyme constructs. The enzyme strand is depicted in black font and the substrate strand in red font. Cleavage sites are shown with an arrow. (A.) Minimal hammerhead ribozyme (HH) derived from Schistosoma mansoni. 1 For the LHH ribozyme enzyme strand (ELHH), long flanking sequences were added to the 5’ and 3’ ends of the minimal HH structure. (B.) HH16 ribozyme used by Uhlenbeck that does not appear to form alternative structures............................................................. 4 Scheme 1: ATPS preparation for partitioning and kinetics studies........................................... 9 Scheme 2: Experimental design for determining the rate of confined hammerheads in ATPS. ................................................................................................................................... 12 Figure 3: Relationship between the concentration of RNA in the dextran-rich phase of an ATPS and log K. These are predicted values calculated based on Equation 7, with the overall RNA concentration held constant at 2 nM. Traces are shown for various volume ratios of dextran-rich:PEG-rich phase. ................................................................. 15 Figure 4: Log of the partitioning coefficient for linear and hairpin DNA in ATPS with different weight percent of PEG 8 kDa and dextran 10kDa. In addition to PEG and dextran the ATPS were made with 100 mM NaCl and 10 mM phosphate buffer (pH 7.5). ....................................................................................................................................... 17 Figure 5: Influence of NaCl concentration on nucleic acid partitioning. Linear and hairpin DNA were partitioned in 10% PEG/12% dextran ATPS with 10 mM phosphate buffer (pH 7.5) and a range of NaCl concentrations. ........................................................ 18 Figure 6: Autoradiogram of a hydrolysis partitioning gel. RNA fragments from the alkaline hydrolysis of ELHH RNA were partitioned in three identical 10% PEG/ 16% Dextran/ 100mM NaCl/ 10mM MgCl2 ATPS. Aliquots from the PEG and dextran phases of the ATPS were fractionated by denaturing PAGE. Lanes 2 – 7: RNA fragments from the dextran-rich phase of the ATPS. Lanes 8 – 13: RNA fragments from the PEG-rich phase of the ATPS. Lanes 5 – 7 and 11 - 13 were loaded 30 min prior to lanes 2 – 4 and 8 – 9 in order to improve band resolution for longer RNA fragments. Lanes 1 and 14 are RNase T1 sequencing ladders for G, assigned to the left of the gel........................................................................................................................
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