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Development of Functional Dna-Based Sensors and Investigations Into Their Mechanism DEVELOPMENT OF FUNCTIONAL DNA-BASED SENSORS AND INVESTIGATIONS INTO THEIR MECHANISM BY NANDINI NAGRAJ DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2010 Urbana, Illinois Doctoral Committee: Professor Yi Lu, Chair Associate Professor Scott Silverman Professor Kenneth Suslick Professor Jonathan Sweedler ABSTRACT The discovery that nucleic acids could perform functional roles in addition to being genetic materials carriers opened doors to a new paradigm in nucleic acid chemistry. Catalytic DNA molecules also known as deoxyribozymes or DNAzymes were first isolated in 1994 through an in vitro selection procedure and have since been engineered and isolated to perform various functions that include both RNA and DNA cleavage and ligation. The 8-17 DNAzyme is an RNA-cleaving DNAzyme that has shown high selectivity for Pb2+ under different selection conditions. It has been explored extensively in terms of its applications for bio-sensing as well as for exploring its mechanism from a more fundamental perspective. A critical barrier of DNA-based sensors for practical applications, such as environmental monitoring, is their highly variable sensing performance with changing temperatures, due to the reliance of sensor design on temperature-dependent hybridization. In Chapter 2, this issue has been addressed through the introduction of mismatches in the DNA hybridization arms of this Pb2+-specific 8-17 DNAzyme and these fluorescent sensors can resist temperature-dependent variations from 4 °C to 30 °C. The strategy of using mismatches to tune the temperature dependence is a novel and inexpensive method that can be applied in other nucleic acid sensors for either metal ions or other molecular targets. Currently there is no structure, (either X-ray or NMR) available for the 8-17 DNAzyme. Hence, understanding its mechanism has posed a challenge, particularly in regard to the high selectivity of Pb2+ for this DNAzyme. In Chapter 3, the systematic activity, folding and structural studies of the 8-17 DNAzyme with both monovalent and divalent metal ions has been carried out. The results obtained suggest a clear trend between the folding and activity of all the metal ii ions studied, the lower the activity, the lesser the folding and vice-versa. Structural studies based on CD and folding studies based on FRET have demonstrated that Pb2+ behaves in a manner that is different from other metal ions and hence it is hypothesized that the 8-17 DNAzyme may have a specific binding pocket for Pb2+. The possibility that the 8-17 DNAzyme might have a metal ion binding pocket for Pb2+ has been investigated in Chapter 4, through systematic phosphorothioate (PS) modifications on the backbone of the DNAzyme. Kinetic assays with the PS modified bases have shown that there are specific bases on the enzyme strand which are important for activity mainly in the presence of Pb2+. The activities of the identical PS modified enzymes are, however, not significantly altered in the presence of Mg2+ and Cd2+. 31P NMR has been used as an additional tool to directly visualize the backbone phosphates since a single PS modification shifts the signal of the phosphate downfield by ~50 ppm. These results, in conjunction, have led to the identification of a proposed metal ion binding site, specifically a potential Pb2+-binding site for the 8-17 DNAzyme. The starting point towards the development of a successful functional nucleic acid-based sensor is its isolation and this is done through in vitro selection. In vitro selection to isolate DNAzymes for Hg2+ and Cd2+ and the use of negative selections to overcome Pb2+ interference at various stages of selection has been described in Chapter 5. Chapter 6 describes the structure- switching strategy to isolate DNA aptamers specific for endotoxins. It is anticipated that the results obtained from the current study and future characterizations will lead to the development of functional DNA sensors for endotoxins. iii Dedicated to my family who have given me their unwavering love and support through everything and who have been the driving force through all the ups and downs. iv ACKNOWLEDGEMENTS I would like to firstly acknowledge my research advisor, Prof. Yi Lu for guiding me through this journey and giving me the freedom to experiment and learn, and providing encouragement when everything seemed to be stumbling along the way. I also would like to thank my committee members Prof. Scott Silverman, Prof. Ken Suslick and Prof. Jonathan Sweedler for their helpful suggestions, particularly during my preliminary examination. Their suggestions and comments added great value to my research and thesis. I would also like to particularly acknowledge the guidance and suggestions provided by Prof. Scott Silverman and his group members, Dr. Elizabeth Pratico and Dr. Dana Baum during trouble-shooting in vitro selection procedures. I also want to thank my collaborator from the Illinois Sustainable technologies Center, Jennifer Deluhery for working with me on the selection of endotoxin aptamers and bringing both her enthusiasm and passion on the table. The secretaries in the IMP office, Connie Knight, Beth Myler, Teresa Struss and Sandy Pijanowski, have really made life extremely smooth for graduate students with their reminders and virtual take-over of all the formalities, and they have really kept us well-fed as well with their wonderful treats and treat-days every month. I also want to acknowledge Dot Gordon who has been a great resource and „go-to‟ person for graduate students and Joyce Beasley for all their help over the years. The Lu lab has been like family and many relationships, both professional and personal that have been formed here will most certainly last for a long time. I want to specially thank Dr. Juewen Liu for pioneering various projects and for great intellectual advice. I also want to thank Dr. Hee-Kyung Kim and Dr. Debapriya Mazumdar for their collaboration on the biochemical v and biophysical characterization of the 8-17 DNAzyme with monovalent metal ions project and thank them as well for valued discussions and for long-lasting support and friendships. I also want to thank Eric Null for all his help on 31P NMR and Seyed-Fakhreddin Torabi for help with the activity assays related to the PS project. I also want to thank undergraduates Stephanie Sterling and Jenny Wu for their help. I want to specially thank Tian Lan and Hannah Ihms for being cubicle buddies and great sources of selection discussions. I also want to thank Weichen Xu, Natasha Yeung and Masha Savelieff for their friendship over the years in the Lu lab. I also want to thank other past and present members of both the protein and DNA lab for sharing their research-related sorrows and joys and for being great people to work with. Finally, I want to thank my family and other friends without whom I wouldn‟t be here today. My parents Appa and Amma, have given me a lot of freedom and have always motivated me to do my best and leave the rest, and encouraged me every step of the way and been my rock of emotional strength and support . My sister Aarts who has also provided me constant emotional support through the years and has always amazed me with her courage and persistence. My parents-in-law, Ma and Baba have encouraged me constantly to be focused and attain my goals. My friends outside of lab, Madhu and Sunni, Gagan and Sukhi, Devi and Inder, Deepa and Adarsh, Preeti and Sandeep, Vikas and Deepti, Sagar and Shweta and Sima have been a lot of fun to hang out with and enjoy time with outside of lab. I finally want to specially thank my husband, Indraneel who has been very supportive of me and my ambitions and shared all the ups and downs, and the joys and the frustrations of my graduate school life and has always been there for me. I humbly acknowledge the Blessings and Grace of Bhagawan Sri Sathya Sai Baba whose love is the constant in my life. vi TABLE OF CONTENTS 1 Introduction ....................................................................................................................... 1 1.1 Nucleic acids .......................................................................................................... 1 1.1.1 Functional nucleic acids ...................................................................................... 1 1.2 Role of metal ion cofactors in catalysis by functional nucleic acid enzymes ... 6 1.2.1 Catalysis based on electrostatic charge screening ............................................... 7 1.2.2 Direct involvement of metal ions in catalysis ..................................................... 8 1.2.3 Indirect involvement of metal ions in catalysis ................................................. 10 1.3 Characterization of functional nucleic acid enzymes ...................................... 10 1.3.1 Biochemical characterization ............................................................................ 10 1.3.2 Biophysical characterization ............................................................................. 12 1.3.3 Crystallographic studies .................................................................................... 12 1.4 Bio-sensing Applications of functional nucleic acids ....................................... 15 1.4.1 Fluorescence-based sensing with DNAzymes .................................................. 15 1.4.2 Fluorescence-based sensing with aptamers ......................................................
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