8-alkoxyadenosines modulate RNA interference efficacy and off-pathway protein binding

Despite the tremendous potential of short interfering RNA (siRNA) as a novel biopharmaceutical, its therapeutic utility has not been maximized mainly due to lack of proper in vivo delivery vehicle, off-target effects and several off-pathway protein interactions instigating immunostimulation. Judicious chemical modification of different parts of the siRNA was foreseen as a potential solution to the off-target gene silencing and the off-pathway protein binding. In this study, 8-alkoxyadenosines were explored as a nucleobase modification in the context of the siRNA-based RNA interference (RNAi). These nucleosides are unusual in that they have the potential to exist as an equilibrium mixture of the syn and anti conformers. When placed opposite to U in the complementary strand, 8- alkoxyadenosines can exist in normal anti conformation and form canonical Watson- Crick hydrogen bonding; interestingly, with G as the base-pairing partner, these unusual nucleosides can potentially flip into the syn conformation and form unorthodox Hoogsteen base-pairing. 8-Alkoxyadenosine phosphoramidites were synthesized and incorporated into the guide strand of caspase 2 siRNA at four different positions - two in the seed region, one at the cleavage junction and another nearer to the 3´-end of the guide strands. Thermal stabilities of the corresponding siRNA duplexes showed that U is still preferred over G as the base-pairing partner in the complementary strand. When compared to the unmodified iv positive control siRNAs, singly modified siRNAs have knocked down caspase 2 insert mRNA (generated from a recombinant plasmid) efficiently and with little or no loss of efficacy. Doubly modified siRNAs were found to be less effective and lose their efficacy at low nanomolar concentrations. Persistent placement of steric blockade in the minor groove affected the RNAi efficacy significantly; this observation supports the hypothesis and indicates the necessity of ‘switching' the bulky alkyloxy groups in the major groove, when modified siRNAs interact with the RISC assembly. SiRNAs modified at positions 6 and 10 of the guide strand were found to be effective against preventing interaction with the RNA-dependent protein kinase (PKR). In summary, 8-alkoxyadenosine-containing siRNAs prevented undesired off-pathway protein binding, without compromising the RNAi efficacy significantly.

8-ALKOXYADENOSINES MODULATE RNA INTERFERENCE EFFICACY AND OFF-PATHWAY PROTEIN BINDING by Uday G hanty A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry University of Utah December 2012 Copyright © Uday Ghanty 2012 All Rights Reserved The Univers i ty of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Uday Ghanty has been approved by the following supervisory committee members: Cynthia J. Burrows , Chair 08/16/12 Date Approved Peter F. Flynn , Member 08/16/12 Date Approved Janis Louie , Member 08/16/12 Date Approved Darrell R. Davis , Member 08/16/12 Date Approved Ilya Zharov , Member 08/16/12 Date Approved and by Henry S. White , Chair of the Department of Chemistry and by Charles A. Wight, Dean of The Graduate School. ABSTRACT Despite the tremendous potential of short interfering RNA (siRNA) as a novel biopharmaceutical, its therapeutic utility has not been maximized mainly due to lack of proper in vivo delivery vehicle, off-target effects and several off-pathway protein interactions instigating immunostimulation. Judicious chemical modification of different parts of the siRNA was foreseen as a potential solution to the off-target gene silencing and the off-pathway protein binding. In this study, 8-alkoxyadenosines were explored as a nucleobase modification in the context of the siRNA-based RNA interference (RNAi). These nucleosides are unusual in that they have the potential to exist as an equilibrium mixture of the syn and anti conformers. When placed opposite to U in the complementary strand, 8- alkoxyadenosines can exist in normal anti conformation and form canonical Watson- Crick hydrogen bonding; interestingly, with G as the base-pairing partner, these unusual nucleosides can potentially flip into the syn conformation and form unorthodox Hoogsteen base-pairing. 8-Alkoxyadenosine phosphoramidites were synthesized and incorporated into the guide strand of caspase 2 siRNA at four different positions - two in the seed region, one at the cleavage junction and another nearer to the 3´-end of the guide strands. Thermal stabilities of the corresponding siRNA duplexes showed that U is still preferred over G as the base-pairing partner in the complementary strand. When compared to the unmodified iv positive control siRNAs, singly modified siRNAs have knocked down caspase 2 insert mRNA (generated from a recombinant plasmid) efficiently and with little or no loss of efficacy. Doubly modified siRNAs were found to be less effective and lose their efficacy at low nanomolar concentrations. Persistent placement of steric blockade in the minor groove affected the RNAi efficacy significantly; this observation supports the hypothesis and indicates the necessity of ‘switching' the bulky alkyloxy groups in the major groove, when modified siRNAs interact with the RISC assembly. SiRNAs modified at positions 6 and 10 of the guide strand were found to be effective against preventing interaction with the RNA-dependent protein kinase (PKR). In summary, 8-alkoxyadenosine-containing siRNAs prevented undesired off-pathway protein binding, without compromising the RNAi efficacy significantly. For my parents TABLE OF CONTENTS ABSTRACT..…………………………………………………………………………….iii LIST OF FIGURES..……………………………………………………………………..ix LIST OF TABLES..……………………………………………………………………..xii LIST OF ABBREVIATIONS..…………………………………………………………xiii ACKNOWLEDGEMENTS..…………………………………………………………...xvii CHAPTERS 1. SHORT INTERFERING RNA THERAPEUTICS AND CHEMICAL MODIFICATI-ONS..……………...………………………………………………………………….. 1 RNA Interference and short interfering RNA...……………………………………1 RNAi: Proof-of-principle in animal models...……………………………………..3 Local RNAi in animal models...…………………………….......................3 Systemic RNAi in animal models...………………………………………..7 SiRNA clinical trials...……………………………………………………………..7 SiRNA delivery...…………………………………………………………………..9 SiRNA therapeutics - pros and cons...…………………………………………...14 Chemical modifications of the siRNA……………………………………………15 Ribose modifications...…………………………………………………...16 Backbone modifications……………………………………......................19 Terminal modifications and bioconjugates...………..……………………22 Base modifications...……………………………………………………...22 Conclusion...……………………………………………………………………...27 References...……………………………………………………..………………..28 2. 8-ALKOXYADENOSINES AS POTENTIAL MODULATORS OF OFF-PATH-WAY PROTEIN BINDING..……..…………………………………………………41 Introduction...…………………………………………………………………….41 Off-target gene silencing...……………………………………………….42 Sequence-specific immunostimulation...…………………………………44 Sequence-independent off-pathway protein binding and immunostimu-lation...……………………………………………………………..……. 44 vii Nucleobase modifications to prevent the siRNA-PKR interaction...……47 Synthesis of the 8-alkoxyadenosine phosphoramidites...……………......52 Experimental...…………………………………………………………………...55 8-Bromoadenosine (1)...………………………………………………...57 5´,3´-O-Bis(-t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-8-bromoadenosine (3)...………………………………………………………………………57 5´,3´-O-Bis(t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-8-propargyloxy-adenosine (4)...…………………………………………………………...58 5´,3´-O-Bis(t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8- propargyloxyadenosine (5)...……………………………………………59 2´-O-t-Butyldimethylsilyl-N6-benzoyl-8-propargyloxyadenosine(6)...…60 5´-O-(4,4´-Dimethoxytrityl)-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8- propargyloxyadenosine (7)...……………………………………………61 5´-O-(4,4´-Dimethoxytrityl)-3´-O-[(2-cyanoethoxy)(N,N-diisopropyl-amino) phosphino]-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8- propargyloxy-adenosine (8)...……………………………………………62 5´,3´-O-Bis(t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-8-phenylethoxy-adenosine (9)...…………………………………………………………...63 5´,3´-O-Bis(t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8- phenylethoxyadenosine (10)...…………………………………………..63 2´-O-t-Butyldimethylsilyl-N6-benzoyl-8-phenylethoxyadenosine (11)....64 5´-O-(4,4´-Dimethoxytrityl)-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8- phenylethoxyadenosine (12)...…………………………………………...64 5´-O-(4,4´-Dimethoxytrityl)-3´-O-[(2-cyanoethoxy)(N,N-diisopropyl-amino) phosphino]-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8-phenyleth-oxyadenosine (13)...……………………………………………………...65 5´,3´-O-Bis(t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-8cyclohexylethoxy-adenosine (14)...………………………………………………………….65 5´,3´-O-Bis(t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8-cyclo-hexylethoxyadenosine (15)…..…………………………………………..66 2´-O-t-Butyldimethylsilyl-N6-benzoyl-8-cyclohexylethoxyadenosine (16)…………………………………..…………………………………...66 5´-O-(4,4´-Dimethoxytrityl)-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8- cyclohexylethoxyadenosine (17)…..…………………………………….66 5´-O-(4,4´-Dimethoxytrityl)-3´-O-[(2-cyanoethoxy)(N,N-diisopropyl-amino) phosphino]-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8-cyclohexyl-ethoxyadenosine (18)…..………………………………………………...67 Design of the caspase 2 siRNA and synthesis of the modified guide Strands...………………………………………………………………….68 Synthesis and purification of siRNAs...………………………………….68 ESI-MS characterization of the modified guide strands...……………….70 Results and discussion...…………………………………………………………70 Conclusion...……………………………………………………………………..76 References...……………………………………………………………………...79 viii 3. THERMAL ANALYSIS AND BIOLOGICAL EVALUATION OF THE MODIFIED SIRNAS..…………………………………………………………………………….82 Introduction...…………………………………………………………………….82 Importance of thermal denaturation analysis of the siRNA...……………82 Consequence of chemical modifications on the melting temperature (Tm) of the siRNAs...…………………………………………………………..83 Influence of thermal stability on the RNAi efficacy.………………….....84 Chemical modifications modulate siRNA efficacy..………………..…...85 Off-pathway protein interactions with the siRNA...……………………..85 Guide strand nucleobase modifications to prevent off-pathway protein binding…………………………………………………………….……..86 Janus-faced 8-alkoxyA nucleosides in the guide strand...………....…….87 Experimental...…………………………………………………………………...89 SiRNA annealing...………………………………………………………89 Thermal analysis of the modified siRNAs...……………………………..90 Synthesis of the wild-type and mutant plasmids ……………………….90 Cell culture...……………………………………………………………..94 The RNAi assay...………………………………………………………..96 The PKR binding assay...………………………………………………...98 Results and discussion...………………………………………………………..100 Thermal analysis of the 8-AlkoxyA-containing siRNAs...……………..100 Caspase 2 mRNA knockdown studies with the modified siRNAs...…...105 Importance of switching the steric blockade from the minor to major groove in the RISC………….…………………………………………..108 The PKR binding studies...……………………………………………..111 Conclusion...……………………………………………………………………118 References...…………………………………………………………………….119 4. CONCLUSION AND FUTURE DIRECTIONS….………………………………..123 LIST OF FIGURES Figure Page 1.1. Structure of an siRNA...……………………………………………………………2 1.2. Mechanism and side effects of the siRNA-based RNAi...…………………………4 1.3. Organs and diseases where siRNA-based RNAi has been demonstrated...………..5 1.4. In vivo delivery vehicles of the siRNA...…………………………………………10 1.5. SiRNA ribose modifications...……………………………………………………17 1.6. SiRNA backbone modifications...………………………………………………..20 1.7. SiRNA terminal modifications...……………………………………………........23 1.8. SiRNA bioconjugates...…………………………………………………………...24 1.9. SiRNA base modifications...……………………………………………………...25 2.1. Ribose modifications to prevent off-target gene silencing...……………………..43 2.2. Ribose modifications to prevent sequence-specific immunostimulation...……….45 2.3. Structure and mechanism of activation of the RNA-dependent protein kinase (PKR)...…………………………………………………………………………...46 2.4. Minor groove purine modifications to prevent PKR binding...……..……………48 2.5. Sites of siRNA passenger strand modifications to prevent PKR binding...……….50 2.6. ‘Switchable' N2-alkylated 2´-deoxy-7,8-dihydro-8-oxoguanosines in duplex RNA to prevent PKR binding: (A) cartoon (B) base-pairs....……………………………...51 2.7. Proposed base-pairing of the 8-alkoxyadenosines…..……………………………53 2.8. Proposed base ‘switch' cartoon to prevent off-pathway protein interaction while x maintaining RNAi efficacy...……………………………………………………...54 2.9. Synthetic scheme of 8-alkoxyA phosphoramidites………….……………….........56 2.10. SiRNA sequences used….………………………………………………………...69 2.11. Degradation of the 8-benzyloxyadenosine derivative……………………………..74 3.1. Proposed 8-AlkoxyA ‘base switches'. (A)Cartoon showing flipping of a steric blockade from the minor to the major groove. (B) Proposed base pairs of the 8- alkoxyAs..….…………………...…………………………………………………88 3.2. psiCHECKTM-2 vector...…………………………………………………………..93 3.3. Caspase 2 inserts with Not I and Xho I restriction sites: (A) wild type (wt), (B) P6 mutant and (C) P10 mutant...……………………………………………………..94 3.4. Synthesis of the recombinant plasmid...…………………………………………..95 3.5. RNAi protocol...…………………………………………………………………...97 3.6. PKR binding experiment protocol...…………………………………………..…..99 3.7. Thermal analysis of the siRNA duplexes with single 8-alkoxyA modifications at four different positions, 4, 6, 10 and 15, in the guide strand...…………………..102 3.8. Thermal analysis of the siRNA duplexes with double 8-alkoxyA modifications at hree different positions, 6, 10 and 15, in the guide strand...…………………..103 3.9. RNAi studies with the singly modified siRNAs...……………………………….106 3.10. RNAi studies with the doubly modified siRNAs...………………………………109 3.11. % Expression of Renilla luciferase relative to the firefly luciferase when treated with siRNAs bearing 8-alkoxyA modifications at positions 6 and 10 opposite to either G or U……………………………………………………………………..110 3.12. RNAi studies on the P6 mutant plasmid (caspase 2 insert containing plasmid mutated at position 6 corresponding to the guide siRNA)...……………………..112 3.13. RNAi studies on the P10 mutant plasmid (caspase 2 insert containing plasmid mutated at position 10 corresponding to the guide siRNA)...……………………113 3.14. PKR binding to the modified siRNAs containing 8-alkoxyadenosine ‘switches'.115 3.14. Typical western blot results for each of the duplexes tested for off-pathway protein- xi binding...…………………………………………………………………………116 4.1. Self-complementary 14-mer RNA duplexes depicting (A) 8-PrNHdA:U and (B) 8- PrNHdA:I combinations...……………………………………………………….129 LIST OF TABLES Figure Page 1.1. SiRNA clinical trials...……………………………………………………………..8 1.2. In vivo delivery strategies for therapeutic siRNAs...……………………………..12 2.1. ESI-MS of the modified guide strands...………………………………………….71 3.1 Caspase 2 siRNAs containing 8-alkyloxyA in the guide strands and U in the complementary passenger strands...………………………………………………91 3.2 Caspase 2 siRNAs containing 8-alkyloxyA in the guide strands and G in the complementary passenger strands...………………………………………………92 xiii LIST OF ABBREVIATIONS A adenosine ADAR adenosine deaminases that act on RNA Ago argonaute AMD age-related macular degeneration Arg arginine 8-BrA 8-bromoadenosine 8-BrdA 2´-deoxy-8-bromoadenosine C cytosine 8-CeOA 8-cyclohexylethyloxyadenosine 8-ClA 8-chloroadenosine dA 2´-deoxyadenosine dC 2´-deoxycytosine dG 2´-deoxyguanosine DMEM Dulbecco's Modified Eagle's Medium DNA 2´-deoxyribonucleic acid dsRB double-stranded RNA-binding dsRBMs double stranded RNA-binding motifs dsRNA double-stranded RNA dT 2´-deoxythymine xiv EBER Epstein-Barr virus-associated encoding small RNA E. coli Escherichia coli EDTA ethylenediaminetetraacetic acid eIF2! eukaryotic initiation factor 2 alpha subunit ESI-MS electrospray ionization mass spectrometry FBS fetal bovine serum G guanosine HRMS high resolution mass spectrometry HPLC high performance liquid chromatography INF Interferon LNA locked nucleic acid miRNA microRNA mRNA messenger RNA NOESY nuclear overhauser effect spectroscopy NMR nuclear magnetic resonance OAS 2´, 5´-oligoadenylate synthase 8-OxodG 2´-deoxy-7,8-dihydro-8-oxoguanosine PAGE polyacrylamide gel electrophoresis PAZ PIWI/Argonaute/Zwille domain PBMC peripheral blood mononuclear cells PCR polymerase chain reaction PDC plasmacytoid dendritic cells 8-PeOA 8-phenethyloxyadenosine xv 8-PgOA 8-propargyloxyadenosine PIWI P-element-induced whimpy testes domain PKR RNA-dependent protein kinase PNA peptide nucleic acid RBP RNA-binding protein RISC RNA-Induced Silencing Complex RLC RISC loading complex RNA ribonucleic acid 8-RNHA 8-alkylaminoadenosine 8-ROA 8-alkoxyadenosine RSV respiratory syncytial virus rtPCR real-time PCR siRNA short interfering RNA ssRNA single-stranded DNA STAT signal transducers/activators TAR HIV-1 transactivation responsive element TLC thin layer chromatography TLR Toll-like receptor TRAP trp RNA-binding attenuation protein TRBP HIV-1 transactivation responsive element SNP single nucleotide polymorphism UNA unlocked nucleic acid UTR untranslated region xvi WC Watson-Crick T thymine Tm melting temperature TMS tetramethylsilane Tris tris(hydroxymethyl)-aminomethane U uracil UV ultraviolet Vis visible ACKNOWLEDGEMENTS I would like to sincerely thank my mentor Professor Cynthia J. Burrows for her continuous support during my doctoral research. She gave me the opportunity to explore synthetic organic chemistry, and the chemistry and chemical biology of RNA in detail. Her patient guidance made this research possible. I also express my sincere gratitude to Professor Burrows for being considerate on many inconveniences that I faced as an international student. I would also like to thank Dr. James G. Muller for running numerous MS samples and helping me technically many times during this study. My gratitude is extended to our collaborator Professor Peter A. Beal for helpful suggestions, and his graduate students Erik Fostvedt and Rachel Valenjuella for performing the PKR binding experiment. I would also like to sincerely thank Dr. Subrata Das and Shrawan Mageswaran for their helpful suggestions in biological experiments. I sincerely acknowledge my committee members Professor Peter F. Flynn, Professor Janis Louie, Professor Darrell R. Davis and Professor Ilya Zharov for their guidance and helpful suggestions during this study. I would specially thank Professor Peter F. Flynn for his help in the structural analysis of the duplex RNAs. I thank the past and present members of the Burrows Laboratory, Dr. Robyn Hickerson, Dr. Yu Ye, Dr. Xiaobei Zhao, Dr. Xiaoyun Xu, Dr. Xin Chen, Dr. Michael Cross, Dr. Aaron Flemming, Dr. Arunkumar Kannan, Khiem van Ngugen, Pranjali xviii Ghude, Na An, Xibo Li, Anna Wolna, Yun Ding, Omar Alsykhly and Lidong He, for their generous help during this study. I express sincere gratitude to my parents, sisters, grandparents, uncles, aunts, cousins and friends for their support and understanding during this study. I am especially grateful to my parents for everything they have done for me; I would not have made it through without their continuous support, love and well wishes. CHAPTER 1 SHORT INTERFERING RNA THERAPEUTICS AND CHEMICAL MODIFICATIONS RNA interference and short interfering RNA RNA interference (RNAi) has become a crucial tool to interrogate and analyze gene function since the discovery of double stranded RNA (dsRNA)-mediated RNAi in C. elegans in 1998 (1). Theoretically, any mRNA in vitro or in vivo can selectively be knocked down by short RNA oligomers containing 19-23 nucleotides called short interfering RNAs (siRNAs). In 2001, Elbashir et al. showed that RNAi was effective in mammalian cells, and the therapeutic potential of siRNA was quickly understood (2). This finding triggered a plethora of research on utilization of siRNA as therapeutics (3-6). SiRNA-mediated gene silencing is highly selective provided siRNAs are rationally chosen. Thermodynamic stability of the duplex is an important determining factor. The duplex should be less stable towards the 5´-end of the guide strand compared to the 3´- end. Nucleotides 2-8 from the 5´-end of the guide strand, also called the seed region of an siRNA (Figure 1.1), play crucial roles in mRNA knockdown efficiency and specificity. Nucleotides 10 and 11 are the cleavage site nucleotides; the mRNA cleavage occurs between these two positions. Generally, there should be perfect base pairing in the seed region and at the cleavage site for optimal efficacy; sometimes, mismatches (7, 8) are tolerated at the expense of RNAi efficacy. 2 Figure 1.1. Structure of an siRNA. (reproduced with permission from reference (9)). 3 SiRNAs can be generated in the cell from long dsRNAs (by Dicer) or can be chemically synthesized and delivered inside cells to mediate specific mRNA cleavage (Figure 1.2). Upon entry into the cellular environment, siRNA is phosphorylated at the 5´-end of the strands and loaded into the RNA-induced silencing complex (RISC). In the RISC, enzyme helicase unwind the siRNA and the active siRNP complex is generated where the guide strand mediates the cleavage of complementary mRNA. RNAi: Proof-of-principle in animal models Local RNAi in animal models. To date, most of the siRNAs have been successfully applied through local administration. Specific silencing of target genes has been achieved in animal models of human diseases using appropriate siRNA or shRNA (short hairpin RNA). The RNAi concept has been validated in multiple organs such as, lung (10, 11), brain (12-20), colon (21, 22), eye (23-25), and vagina(26) and diseases including viral infection (10, 11, 27, 28), nervous diseases (12-16, 29, 30), cancers (22, 31-36), irritable bowel syndrome(21), etc.(37) (Figure 1.3). Highly promising results have been obtained in rodent (mouse) and primate (monkey) models of severe acute respiratory syndrome (SARS) (11), RSV (respiratory syncitical virus) and influenza. The cationic polymer-based transfection reagent TransIT TKO has successfully led to intranasal delivery of siRNAs against RSV and para-influenza virus into pulmonary tissues and reduced the infection by more than 99% (10). Lethal Ebola virus infection was successfully countered by administration of SNALP-formulated siRNAs in guinea pigs (28). RNAi proof-of-concept studies have been demonstrated in numerous ocular diseases, in animal models, through local intravitreal delivery of specific siRNA. SiRNAs 4 Figure 1.2. Mechanism and side effects of the siRNA-based RNAi. P P Dicer Kinase RISC loading complex P P PIWI PAZ HELICASE Ago2 Active siRNP in the RISC siRNA unwinding P P PIWI PAZ Ago2 Active siRNP in the RISC mRNA recognition, seeding and nucleation mRNA Cleavage Off-pathway protein binding Sequence independent Sequence dependent PKR TLR7 TLR8 Immunostimulation TRBP P P PIWI PAZ Ago2 5 Figure 1.3. Organs and diseases where siRNA-based RNAi has been demonstrated. (redrawn with permission from reference (37). Direct RNAi Systemic RNAi Lung RSV Flue SARS Eye AMD (wet) Nervous system Depression Alzheimer disease Huntington disease Tumor Glioblastoma Human papillomavirus Prostate Lung Influenza Spinocerebral ataxia ALS Neuropathic pain Encephalitis, West Nile virus Adenocarcinoma Digestive system Irritable bowel syndrome Vagina HSV Tumor Liver Joint Rheumatoid arthritis Hypercholesterolemia HBV 6 targeting vascular endothelial growth factor (VEGF) receptor-1 and transforming growth factor (TGF)-β receptor type II were effective in shrinking ocular neovascularization and preventing choroidal neovascularization respectively in two separate mouse models (23, 24). For local siRNA delivery in the eye, saline and lipid formulations were proven effective. Mucosal membranes in colon, nose and genitals are accessible to various siRNA formulations, e.g., lipofectamine formulated siRNAs targeting TNF-α in IBS has been shown to reduce TNF-α abundance, as well as reduce inflammation of the colon (21). ShRNA- or siRNA-based RNAi was shown to be effective in vivo in numerous nervous disease models (12-16, 29, 30). RNAi has brought about marked reduction in disease phenotypes and neuropathology (16) in the xenograft models of Alzheimer's disease (16), Huntington's disease (13), amyotrophic lateral sclerosis (ALS) (14, 15), chronic neuropathic pain (38), cerebellar ataxia (12), encephalitis (30), etc. In these studies on specific neurons, even 10-20% reduction of the target gene expression has been reported to normalize disease symptoms significantly (16). The RNAi concept has also been validated in different mouse xenograft models of human cancers through appropriate delivery vehicles (37), such as lipids (31, 39, 40), polymers (41), cholesterol-oligoarginine (22), protamine-Fab fusion protein (42), and aptamers (43). Aptamer-based chimeric siRNAs promise to be the most simplified and selective approach, provided an aptamer is available for a tumor specific receptor. Aptamer-siRNA conjugates have successfully prevented tumor growth in xenograft models of prostate cancer (34). 7 Systemic RNAi in animal models. In systemic administration of siRNA, RNAi efficacy is dependent on the choice of delivery vehicles and chemical modifications. Systemic RNAi (44, 45) has been demonstrated in mouse models of several diseases such as hypercholesterolemia (46), rheumatoid arthritis, viral infections (hepatitis B virus (27) , influenza virus, Ebola virus (28)) and in tumor xenografts. Cholesterol-conjugated and SNALP (stable nucleic acid lipid particle)-formulated siRNAs have been shown to silence apolipoprotein apoB in nonhuman primates (47). A spectrum of delivery vehicles has successfully delivered anticancer siRNAs into various malignant tumors. Cationic cardiolipin liposome formulated siRNAs, when delivered intravenously, prevented tumor growth in xenograft model of prostate cancer (48). Systemic administration of PEGylated PEI-Arg-Gly-Asp nanoparticle formulated siRNAs targeting VEGF R-2 (vascular endothelial growth factor receptor-2) has prevented tumor angiogenesis and growth (49). Cholesterol-conjugated siRNAs targeting VEGF receptor inhibited tumor growth in colon adenocarcinoma (22). Intravenous delivery of atelo-collagen-siRNA complexes have pronounced effects on bone tumors (35) and subcutaneous tumor xenografts (50). Ligand-directed systemic delivery of siRNA is gradually gaining importance; siRNA complexed with transferrin-anchored polymeric nanoparticles has shrunk metastatic Ewing sarcoma (36). SiRNA clinical trials In spite of the challenges of finding appropriate delivery vehicles for siRNA, at least eleven siRNAs are currently in clinical trials (Table 1.1). Validation of the siRNA-based RNAi in xenograft models of human diseases has encouraged clinical trials of siRNAs in the treatment of several diseases (3, 51, 52). Prior success with antisense oligo 8 Table 1.1 SiRNA clinical trials. (reproduced with permission from reference (6)) 9 nucleotides, led to initial clinical trials of siRNAs targeting ocular diseases such as AMD and diabetic macular edema (DME). In both the cases VEGF pathway was targeted locally with intraocular injection of siRNA. After initial clinical trials both were withdrawn from the market due to safety reasons and availability of alternative therapeutics (53). Still, the eye is an attractive target for RNAi validation (54, 55). SiRNA (PF-04523655) targeting hypoxia-responsive gene RTP801 to treat AMD and DME is already in advanced clinical trial (phase II) (55); visual acuity is restored in 90% of the patients in a preliminary study. Two other drugs for the treatment of glaucoma are currently under phase I clinical trial (6). Due to high efficacy and specificity of siRNAs against RSV in mice models, Alnylam has designed ALN-RSV01 siRNA, which is currently in phase II clinical trial (56). Two siRNAs, for acute renal failure and asthma, are under phase II clinical trial at the moment (6). There has been significant advancement in the development of siRNAs targeting cancer. Several targets for siRNA therapeutics (under phase I clinical trial) in this category are ribonucleotide reductase (RRM2) for solid tumors, PKN3 for tumor vasculature in solid tumors, VEGF and KSP for liver cancer etc. Tekmira is developing lipid nanoparticle-formulated siRNAs targeting ApoB for hypercholesterolemia (6). All of them are being developed for systemic administration. Transderm is developing siRNAs against pachyonychia congenita, a keratin 6a abnormality; they have successfully targeted a single-point mutant mRNA corresponding to keratin 6a (57, 58). SiRNA delivery SiRNA delivery techniques have improved significantly over the last few years (Figure 1.4) (59). Polymeric or liposomal formulations reduced the dose requirement (per 10 Figure 1.4. In vivo delivery vehicles of the siRNA. (adapted with permission from reference (3)) Cholesterol conjugates Transferrin-cyclodextrin polycation nanoparticles SNALPs Aptamer-siRNA chimeras MEA-dynamic polyconjugate particles Positively-charged antibody conjugates transferrin polycation nanoparticles antibody cholesterol 11 day) of naked siRNA in efficacious RNAi; compared to 0.4 mg of siRNA formulated in PBS, only 5-40 μg is required if formulated in appropriate polymeric or liposomal delivery vehicles. Reduction in dosage also helps reduce undesired off-target effects and immunostimulation. Also in ocular clinical trials, it was found that only cholesterol-conjugated siRNAs can enter the RNAi pathway and naked siRNAs merely interact with the innate immune system (60). These observations clearly suggest that appropriate delivery vehicles are essential for therapeutic efficacy of siRNA. Polymeric nanocarriers are gradually gaining importance as siRNA delivery vehicles (41, 61-64). PEG-based and pH sensitive cationic copolymers (65), lactose-conjugated pH-sensitive PEG (66) and chitosan-polyethylene oxide copolymer (67) have been shown to enhance intracellular gene silencing. Acid-lability and bioreduciblity of polymer backbones have been judiciously used for the controlled release of siRNA in desired targets (62, 66, 68). Recently, phosphonium ion-based cationic polymers were employed as a nontoxic alternative to the polyammonium carriers (69). Several macroscopic biomaterial scaffolds, such as PEG, alginate, photoalginate, collagen etc, have been reported for localized, targeted and sustained delivery of siRNA (63, 70). Proton sponge-anchored quantum dots have been used both to deliver siRNA to appropriate target and image the entire delivery and trafficking process inside cell (71, 72). Lipid-based delivery vehicles are also being successfully used for in vivo RNAi studies (Table 1.2) (39, 40, 73). SiRNAs-conjugated to lipid-like molecules have successfully delivered siRNAs in cultured cells and in vivo. These covalently conjugated lipidoids are promising for both local and systemic delivery of siRNA (74). 12 Table 1.2. In vivo delivery strategies for therapeutic siRNAs. (reproduced with permission from reference (75)) 13 Cholesterol-conjugated siRNAs have shown to improve RNAi efficacy significantly both in cultured cells and in vivo (46, 76, 77). SiRNA conjugated to a single wall carbon nanotube efficiently delivered siRNA in cultured HeLa cells and lead to potent silencing of the lamin A/C gene (78). To enhance the therapeutic potential of siRNA and reduce toxicity, targeted delivery is desired and required. Targeted delivery of siRNAs has been accomplished by anchoring an appropriate ligand (recognizable and taken up by a particular type of cell) to the delivery vehicle. Transferrin-anchored polycationic nanoparticles can selectively deliver siRNA into targeted cells by virtue of recognition of transferrin by specific receptors on the target cell surface (79). Folic acid (80, 81) or a specific carbohydrate,(66, 82) when anchored to PEG oligomers, can direct the delivery of siRNAs to selective target or diseased cells, because many cells overexpress folate or carbohydrate receptors under abnormal conditions. PEG-decorated liposomes (SNALPs) can selectively deliver siRNAs in the livers of non-human primates; in one case the RNAi efficacy is >90% and one dose lasts for 11 days, indicating prolonged half-life of the siRNA in that formulation (47). Conjugation of cell-penetrating peptides with siRNAs has also helped reduce dosage and improve RNAi efficacy (83). SiRNAs, when conjugated to liposomes, antibodies or neuropeptides can overcome the blood-brain barrier and can be delivered into the brain (3, 84). Antibody (targeting HIV 1 envelope protein gp160)-conjugated protamines can bind siRNAs (targeting HIV gene gag) electrostatically, and deliver siRNAs inside an HIV-infected murine model (42). Aptamers are gradually gaining popularity as delivery vehicles for the siRNA (85, 86). Aptamers are in vitro-evolved 14 synthetically prepared nucleic acid oligomers that can bind selectively and tightly to specific ligands; therefore aptamers can help the intracellular delivery of other biomolecules through selective aptamers-ligand interactions, provided an aptamer is available for the specific ligand (receptor or a part of the receptor) on the cell-surface. Prostate-specific membrane antigen-binding aptamers when conjugated to siRNAs targeting pro-survival genes in prostate cancer cells have been found to deliver the RNAi agent efficiently inside cells (43, 87). SiRNA therapeutics - pros and cons SiRNA enjoys certain distinctive advantages over small molecule and protein based drugs. Small molecules have the limitation of being useful to "druggable" targets and the application of protein-based therapeutics is restricted to extracellular targets only; contrarily, RNAi can be extended to any biological target, including so called "non-druggable" targets. Like protein-based antibodies and unlike small molecules, siRNAs are very selective and potent. It is much easier to synthesize short RNA oligomers than synthesizing complex organic molecules of biological significance; siRNA lead optimization is also much faster compared to protein-based drugs. In spite of all these advantages of siRNA over other therapeutics, the therapeutic potential of RNAi has not been fully exercised yet because of numerous problems associated, such as ineffective delivery, poor nuclease-stability and numerous undesired side effects. SiRNA molecules, unlike small molecules, have high negative charges in its backbone and hence cannot cross the cellular membrane without the aid of delivery vehicles. Either covalent conjugation with other groups or noncovalent assembly with other materials is essential for their intracellular delivery. 15 Optimal thermal and nuclease stability is essential for the therapeutic efficiency of siRNAs. Double stranded RNAs are much more stable compared to the single stranded ones toward nucleases. Generally, siRNAs are designed so that the duplex is of intermediate thermal stability - if the stability is very high, the RNAi machinery might not be able to unwind the siRNA for selective mRNA knock down; again, if the duplex is unstable (i.e., the melting temperature is low), then the siRNA will be an ineffective one since higher percentage of siRNA will exist as single strands which will be rapidly degraded by serum ribonucleases. Several chemical modifications have been reported to improve the thermal and the nuclease stability of siRNAs and these modifications do not affect the RNAi efficacy too much. Native siRNAs can have off-target effects and instigate immunostimulation; these lower the specificity of siRNA action and induce toxicity inside cells. Several types of off-target effects can arise with siRNA-based therapeutics. Perfect complementarity of various regions of multiple mRNAs with the ‘seed' region of an siRNA is a possible source of undesired side effect. To overcome this problem, several siRNAs are chosen as potential candidates and the one with optimal silencing efficacy and minimal ability to instigate non-target gene repression is chosen as the most promising compound. Judicious chemical modifications are another alternative to achieve RNAi selectivity and avoid undesired repression of non-target gene expression. Chemical modifications of the siRNA Analogous to the well-known antisense technology, chemical modifications of siRNA have promise in improving siRNA properties such as enhanced duplex stability, improved nuclease resistance, higher potency, higher specificity etc. Also, chemically 16 modified siRNAs have facilitated cellular uptake, enhanced half-life, improved biodistribution and better pharmacokinetics. Naked siRNAs are rapidly degraded and excreted through the urinary system. Therefore, suitable chemical modifications are required for the translation of siRNA into potent therapeutics; in fact a large proportion of the siRNAs currently in clinical trial are chemically modified (6). SiRNA modifications can be classified into several categories: ribose modifications, backbone modifications, base modifications and terminal modifications including conjugation to other biologically significant molecules. Ribose modifications. Ribose modifications are the most explored chemical modifications of the siRNA; from subtle to extensive modification of the sugar moiety have been reported (Figure 1.5). Modifications of the ribose moiety (88-105) have improved siRNA stability (88, 96), ribonuclease resistance (88, 90, 93, 95, 99), potency (90, 95, 104), and specificity (88, 106). 2´-OH of the sugar moiety has been modified more frequently than any other positions. 2´-deoxy (94), 2´-F (90) or 2´-N3 (97) substitutions did not significantly affect RNAi efficacy. 2´-Deoxy modifications lower the Tm of the siRNAs slightly, but are tolerated at the terminal of each strands and at the overhangs; however substitution of one of the strands with 2´-deoxy nucleosides (i.e., RNA:DNA hybrid) completely abolishes the RNAi activity (94, 107). 2´-NH2 substitutions maintains RNAi activity, but to a lower extent than the unmodified siRNAs (94). 2´-NH2 modifications led to reduction in RNAi efficacy when placed in the passenger strand and drastic reduction in efficacy when placed in the guide strand (94). In earlier studies, 2´-OMe substitutions (89) have been reported to drastically reduce RNAi effects. Fully 2´-OMe-substituted nucleoside containing siRNAs were not 17 Figure 1.5. SiRNA ribose modifications. O O O O Base O O F O Base O O O O Base O O O Base S O OH O Base S O O O Base S O F O Base O O O O Base O O O O Base O O O O Base O O O O Base O O OH O Base O O O Base F O S O O Base F O O O Base O CN O O O O Base NH3 HO O O O Base O O O Base OH 2'-fluoro 2'-methoxy 2'-MOE 2'-FANA 4'-thio Me-SRNA FSRNA 4'S-FANA LNA ENA OXE ALN ANA HNA 2'-EA 2'-CE HM UNA O O NH O Base AENA O O NH2 O Base 2'-amino O O Base OH O O O Base H2N HA CeNA O O N3 O Base 2'-azido 18 functional, however two to four modifications in either strand were well tolerated. When every other nucleoside was modified with 2´-OMe, siRNAs were significantly resistant to serum nucleases (108). In another study, alternate placement of 2´-F and 2´-OMe modifications both in the guide and the passenger strands increased therapeutic efficacy of the siRNA (90). 2´-MOE (methoxyethyl) modifications were tolerated in the guide strand and alternate placement of 2´-MOE and 2´-OH or several 2´-MOE at the termini improved the RNAi activity (109). In general, the activity of 2´-sugar modifications in siRNAs depends on their size and position in the duplex; small modifications such as 2´- F are generally well tolerated in the guide strand regardless of their position (92, 96). Larger 2´-OMe modifications are well tolerated at the 3´-end of the guide strands and at any positions in the passenger strands. Bulky 2´-MOE modifications are better placed in the passenger strands, whereas their inclusion in the guide strand generally decreases RNAi efficacy (92). 2´-F (88) and locked nucleic acid (LNA) (88) (containing a methylene bridge between the 2´ and the 4´ positions of the ribose) modifications are now frequently used to enhance nuclease resistance, thereby increasing the potency and decreasing the dosage of siRNA. 2´-F, LNA and ENA (ethylene-bridge nucleic acid) are known to restrict the sugar conformation and thereby enhance the specificity of complementary strand recognition and the strength of target binding. These two modifications, along with the 2´-F modification can increase the melting temperature (Tm) of siRNAs significantly (88). SiRNAs containing LNAs at the 5´-end 3´-end or both in the same strand can exhibit RNAi activity (88); however, LNA substitution at the central region of the siRNA leads to complete loss of activity. 19 Substitution of the 4´-O with S has enhanced the Tm and the nuclease resistance of siRNA; 4´-S modified nucleosides are well tolerated at the termini of the both passenger and guide strands (110, 111). 4´-thio-2´-fluoroarabinonucleic acid (4´-S-2´-FANA) modifications, when placed into siRNAs, were able to mediate gene silencing through RNAi pathway; modifications at several sites afforded highly potent siRNAs (98). Alternate placement of 2´-FANA (2´-fluoroarabinonucleic acid) and 2´-FRNA (2´- fluororibonucleic acid) or LNA in an siRNA enhanced its potency (104). Recently the unlocked nucleic acid (UNA)-based sugar modifications were reported; these unrestrained groups can prevent sequence-dependent off-target effects at the expense of slight loss of efficacy (106). Few modifications of the sugar moiety have been reported in which the entire ribose group have been replaced by other groups as in the cyclohexenyl nucleic acid (CeNA), where one or two ribose moieties were replaced with cyclohexene moieties; resultant siRNAs retained their RNAi activity (112). Backbone modifications. Phosphodiester backbone (88, 89, 94) modifications have improved several properties of the siRNA and made it more diverse as a therapeutic agent. Subtle phosphodiester backbone modifications, such as phosphorothioate (PS) and boranophosphate (BP) modifications, as well as complete replacement of the natural backbone with peptide or morpholino modifications have been reported (Figure 1.6). PS modifications are known to improve the ribonuclease resistance and pharmacokinetic profiles of the antisense oligonucleotides (113); these modifications are also known to bind with serum proteins and thereby enhance the half-life of the PS-modified antisense oligomers in the systemic circulation (114). Similar property enhancements might also be observed for siRNAs. However PS-modifications in the passenger strand or in the guide 20 Figure 1.6. SiRNA backbone modifications. O O OH O P O S Base O O OH O Base O O OH O P O BH3 Base O O OH O Base NH N O Base NH N O Base N O O Base O P N O N O Base phosphorothioate (PS) boranophosphate PNA morpholino 21 strand lowers the Tm of the duplex (88) and reduces the RNAi efficacy (89) moderately. Fire and coworkers reported that phopsphorothioate substitution in both the guide and the passenger strands maintain RNAi effect in Caenorhabditis elegans (94). This reduction in RNAi potency may be associated with reduced binding interaction between the PS-modified siRNAs and the RISC complex (100). Extensive substitution of the backbone with PS group has been proven to be cytotoxic; however moderate numbers of PS modifications have been tolerated in siRNAs (7). In the boranophosphate modifications, one of the nonbridging oxygens is replaced with a borane (BH3) group. Enantiomerically pure boranophospate-substituted (BP) siRNAs are more nuclease resistant than racemic PS-substituted or unmodified siRNAs. BP modification slightly increases the Tm of the siRNA duplex and BP-modified siRNAs are more potent than PS-modified siRNAs. The BP-backbone is more lipophilic than the unmodified siRNAs and hence can have significant improvement in the pharmacokinetic and pharmacodynamic profiles of the BP-modified siRNAs compared to the unmodified ones. This modification is better tolerated in the passenger strand than in the guide strand and towards the termini of the oligonucleotides than the central region of the siRNA (115, 116). In peptide nucleic acids (PNAs), the phosphodiester backbone is replaced by a peptide backbone. SiRNA-PNA chimeras at, or in place of the 3´ overhangs, have increased resistance to exonuclease digestion, siRNA half-life and RNAi efficacy (117). Morpholino (118) and triazolyl (119) modifications in those positions afforded similar results. 22 Terminal modifications and bioconjugates. Polymer- (66, 82, 120), lipid- (121), cholesterol- (76, 122), carbohydrate- (66, 82), peptide- (123) and small molecule- (122) based terminal modifications and delivery systems of siRNAs exhibited effective RNAi; polymer- and cholesterol-based conjugates facilitated intracellular delivery of siRNA both in vitro and in vivo (46, 124) (Figures 1.7 and 1.8). For target specific delivery of the siRNA, suitable ligands (e.g., folic acid (81), mannose (82), hyaluronic acid etc.) are chemically attached at the termini of the strands. Small lipid-like molecules (lipidoids) when terminally attached to siRNAs can mediate RNAi in cultured cells and in animal models without the necessity of external delivery vehicles (74, 77). Base modifications. SiRNA base modifications are relatively less explored compared to ribose modifications or terminal modifications because preservation of necessary H-bonding interactions and stable A-form duplex are crucial in effective RNAi (Figure 1.9) (9, 89, 125-131). Disruption of necessary H-bonding interactions (e.g. in N3- Me-U) or steric occlusion of siRNA-RISC interactions (5-IU) can adversely affect the RNAi efficacy and specificity (89). For this reason, most of the successful base modifications have been introduced into either passenger strands or at the 3´-end of the guide strands (125, 129, 131). Base modifications have some additional advantages that cannot be matched by other chemical modifications; the importance of base pairing (126) at certain location in the siRNA can only be explored by suitable base modifications. Similarly, sterically demanding base modifications (e.g., triazolyl-containing click adduct) are tolerated at sites where even a small ribose modification (e.g., 2´-OMe) abolishes the RNAi activity completely (132). 23 Figure 1.7. SiRNA terminal modifications. O O MeO Base 5'-Methoxy OH O O Base 3'-ddc O O O Base 3'-aminomodifier OH P O O O H3N O O O Base 3'-aminomodifier OH P O O O HN O NH O NH H HN H S O O O O Base 3'-aminomodifier OH P O O O HO O O O Base OH O O Base OH O O HN N N R N n n = 1 or 3 H2 N NH 2 H3N O NH O R1 O OH R1 NH2 N N N N N N NH2 NH2 24 Figure 1.8. SiRNA bioconjugates. O O O Base OH O O O Base OH O OH HO OH O OH O OH OH OH O OH NH O S O O S n O CH3 6 O O O Base OH CH2 6 NH O HN D(Cys - Ser - Lys - Cys) KKWKMRRNQFWIKIQR Cys NH2 S O O S Base OH GWTLNSAGYLLKINLKALAALAKKIL Cys NH2 S O O S Base OH O OH HO OH O OH O HO HO OH O O O O O Base P OH O O O S S O (OH2CH2C)n NH O O O O Base P OH O O O S S O (OH2CH2C)n NH O P O O O O OH NH O NH N O HN H2N N N O HN NH P O O 25 Figure 1.9. SiRNA base modifications. NH N O O R 5-Substituted uridine R = I , Br R = CH3 R = C C CH3 NH N O O Dihydrouridine NH N O S 2-Thiouridine HN NH O O Pseudouridine N N O O 3-Methyluridine N N NH2 O 5-Methylcytidine R1 R2 R3 R4 2,4-Difluorotoluene CH3 F H F 2,4-Difluorobenzene H F H F 2,4-Dichlorobenzene H Cl H Cl 2,3-Dichlorobenzene H H Cl Cl R1 R2 R3 R4 N N 4-Methylbenzimidazole NH N N N O NH N N O NH2 N N N N N N R6 NH N N HN O N N N N N NH2 NH N N N O N Hypoxanthine 7-Deazaguanine NO2 5-Nitroindole N N O O NO2 O4-[2-(2-nitrophenyl)propyl]thymidine R5 O NO2 O6-[2-(2-nitrophenyl)propyl]guanine O H R10 H R9 R5 R6 Purine H H 2-Aminopurine H NH2 2,6-Diaminopurine NH2 NH2 N2-Alkyl-2-aminopurines H NH-R7 R7 NH NH NH NH N N N R8 HN O OH OH OH NH HO O R8 = N2-Alkyl-guanines 6-Phenylpyrroloylcytidine R9 = Cyclopentyl, Bn N2-Alkyl-8-oxoguanines R10 = H, Pr, Bn N N NH O 26 Effect of H-bonding on siRNA activity was explored by utilizing nucleobase isosteres in place of natural bases (Figure 1.9) (126, 127, 133). These moieties are hydrophobic and incapable of forming hydrogen bonds with natural nucleobases. Two hydrophobic isosteres of U, 2,4-difluorobenzene and 2,4-dichlorobenzene, when placed in different regions of the guide strand, enhanced thermal stability and maintained moderate RNAi efficacy when placed in the terminal regions of the siRNA. Similarly, 2,4-difluorotoluene-containing siRNAs have enhanced nuclease stability and comparable RNAi activity (127). These modifications are well tolerated at position 7 of the guide strand, however when placed next to the mRNA cleavage site (position 10 or 11) RNAi efficacy is largely abolished (126, 127). 2,3-Dichlorobenzene and 4-methylbenz-imidazole, even though unable to form H-bonds with other nucleosides, prefer U as the complementary base in the mRNA; steric similarity of those modifications with A might be a plausible explanation for this observation (133). Mayer and coworkers reported that introduction of thermally destabilizing groups such as, 2,4-difluorotoluene, hypoxanthine, 5-nitroindole, purine, and 2-aminopurine at various positions within the passenger strand improved RNAi efficacy mainly due to thermal destabilization of the duplex (134). RNA major groove-modulating alkyl groups (methyl and propargyl) when substituted in U or C enhanced the thermal and nuclease stability of the siRNAs; however their effect on RNAi was dependent on the position and size of the modification (128). 2-Thiouracil or pseudouracil at the 3´-terminal of the guide strand increases the RNAi efficacy when a single dihydrouracil is also placed at the 3´-end of the passenger strand (135); this enhancement in efficacy can be attributed to increased duplex asymmetry and facilitated strand unzipping from the 5´-end (135). 27 6-Phenylpyrrolocytosine, a highly fluorescent nucleoside analog, when placed in the passenger strand or towards the 3´-terminal of the guide strand, silenced target genes efficiently and helped visualize intracellular trafficking (131). Minor-groove-modulating guanine and 2-aminopurine modifications have been reported to reduce off-pathway protein interaction (125, 129, 130). Mikat and Heckel investigated the use of the photolabile 2-(2-nitrophenyl)propyl group as a temporary major groove modification of U or G in the guide and passenger strands of the siRNA; but those modifications were found to be fully effective only at a single site (136) . Conclusion The therapeutic potential of siRNA has not yet been exercised fully, mainly due to lack of appropriate delivery vehicles and numerous off-target effects. To address limitations of siRNA, chemical modifications of different segments of the siRNA are a potential solution. Although ribose modifications are most extensively studied, backbone and terminal modifications have also been explored. Terminal modifications of siRNA strands have served multiple purposes, e.g., intracellular delivery, enhancement of siRNA half-life, improving bio-distribution and pharmacokinetic profiles. Although not as common, successful base modifications have been reported both in the passenger and the guide strands. Base modifications provide valuable information regarding necessity of base pairing at certain regions of the siRNA, the scope of modification as applied to A, U, G, and C, the structure of the major and minor grooves during delivery and in the RISC and prevention of off-pathway protein binding. 28 References 1. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391, 806-811. 2. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature 411, 494-498. 3. Castanotto, D., and Rossi, J. J. (2009) The promises and pitfalls of RNA-interference- based therapeutics, Nature 457, 426-433. 4. Dykxhoorn, D. M., and Lieberman, J. (2006) Knocking down disease with siRNAs, Cell 126, 231-235. 5. de, F. A., Vornlocher, H.-P., Maraganore, J., and Lieberman, J. (2007) Interfering with disease: a progress report on siRNA-based therapeutics, Nat. Rev. Drug Discov. 6, 443-453. 6. Watts, J. K., and Corey, D. R. (2010) Clinical status of duplex RNA, Bioorg. Med. Chem. Lett. 20, 3203-3207. 7. Amarzguioui, M., Holen, T., Babaie, E., and Prydz, H. (2003) Tolerance for mutations and chemical modifications in a siRNA, Nucleic Acids Res. 31, 589- 595. 8. Du, Q., Thonberg, H., Wang, J., Wahlestedt, C., and Liang, Z. (2005) A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites, Nucleic Acids Res. 33, 1671-1677. 9. Peacock, H., Kannan, A., Beal, P. A., and Burrows, C. J. (2011) Chemical modification of siRNA bases to probe and enhance RNA interference, J. Org. Chem. 76, 7295-7300. 10. Bitko, V., Musiyenko, A., Shulyayeva, O., and Barik, S. (2005) Inhibition of respiratory viruses by nasally administered siRNA, Nat. Med. 11, 50-55. 11. Li, B.-j., Tang, Q., Cheng, D., Qin, C., Xie, F. Y., Wei, Q., Xu, J., Liu, Y., Zheng, B.-j., Woodle, M. C., Zhong, N., and Lu, P. Y. (2005) Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque, Nat. Med. 11, 944-951. 12. Xia, H., Mao, Q., Eliason, S. L., Harper, S. Q., Martins, I. H., Orr, H. T., Paulson, H. L., Yang, L., Kotin, R. M., and Davidson, B. L. (2004) RNAi suppresses 29 polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia, Nat. Med. 10, 816-820. 13. Harper, S. Q., Staber, P. D., He, X., Eliason, S. L., Martins, I. H., Mao, Q., Yang, L., Kotin, R. M., Paulson, H. L., and Davidson, B. L. (2005) RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model, Proc. Natl. Acad. Sci. USA 102, 5820-5825. 14. Ralph, G. S., Radcliffe, P. A., Day, D. M., Carthy, J. M., Leroux, M. A., Lee, D. C. P., Wong, L.-F., Bilsland, L. G., Greensmith, L., Kingsman, S. M., Mitrophanous, K. A., Mazarakis, N. D., and Azzouz, M. (2005) Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model, Nat. Med. 11, 429-433. 15. Raoul, C., Abbas-Terki, T., Bensadoun, J.-C., Guillot, S., Haase, G., Szulc, J., Henderson, C. E., and Aebischer, P. (2005) Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS, Nat. Med. 11, 423-428. 16. Singer, O., Marr, R. A., Rockenstein, E., Crews, L., Coufal, N. G., Gage, F. H., Verma, I. M., and Masliah, E. (2005) Targeting BACE1 with siRNAs ameliorates Alzheimer disease neuropathology in a transgenic model, Nat. Neurosci. 8, 1343- 1349. 17. Thakker, D. R., Natt, F., Hüsken, D., Maier, R., Müller, M., van der Putten, H., Hoyer, D., and Cryan, J. F. (2004) Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference, Proc. Natl. Acad. Sci. USA 101, 17270-17275. 18. Thakker, D. R., Natt, F., Husken, D., van der Putten, H., Maier, R., Hoyer, D., and Cryan, J. F. (2005) siRNA-mediated knockdown of the serotonin transporter in the adult mouse brain, Mol. Psychiatry 10, 782-789. 19. Luo, M.-C., Zhang, D.-Q., Ma, S.-W., Huang, Y.-Y., Shuster, S., Porreca, F., and Lai, J. (2005) An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons, Mol. Pain 1, 29. 20. Nakajima, H., Kubo, T., Semi, Y., Itakura, M., Kuwamura, M., Izawa, T., Azuma, Y. T., and Takeuchi, T. (2012) A rapid, targeted, neuron-selective, in vivo knockdown following a single intracerebroventricular injection of a novel chemically modified siRNA in the adult rat brain, J. Biotechnol. 157, 326-333. 21. Zhang, Y., Cristofaro, P., Silbermann, R., Pusch, O., Boden, D., Konkin, T., Hovanesian, V., Monfils, P. R., Resnick, M., Moss, S. F., and Ramratnam, B. (2006) Engineering mucosal RNA interference in vivo, Mol. Ther. 14, 336-342. 30 22. Kim, W. J., Christensen, L. V., Jo, S., Yockman, J. W., Jeong, J. H., Kim, Y.-H., and Kim, S. W. (2006) Cholesteryl oligoarginine delivering vascular endothelial growth factor siRNA effectively inhibits tumor growth in colon adenocarcinoma, Mol. Ther. 14, 343-350. 23. Shen, J., Samul, R., Silva, R. L., Akiyama, H., Liu, H., Saishin, Y., Hackett, S. F., Zinnen, S., Kossen, K., Fosnaugh, K., Vargeese, C., Gomez, A., Bouhana, K., Aitchison, R., Pavco, P., and Campochiaro, P. A. (2005) Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1, Gene Ther. 13, 225- 234. 24. Reich, S. J., Fosnot, J., Kuroki, A., Tang, W., Yang, X., Maguire, A. M., Bennett, J., and Tolentino, M. J. (2003) Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model, Mol. Vis. 9, 210- 216. 25. Ahmed, Z., Kalinski, H., Berry, M., Almasieh, M., Ashush, H., Slager, N., Brafman, A., Spivak, I., Prasad, N., Mett, I., Shalom, E., Alpert, E., Di Polo, A., Feinstein, E., and Logan, A. (2011) Ocular neuroprotection by siRNA targeting caspase-2, Cell Death Dis. 2, e173. 26. Palliser, D., Chowdhury, D., Wang, Q.-Y., Lee, S. J., Bronson, R. T., Knipe, D. M., and Lieberman, J. (2006) An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection, Nature 439, 89-94. 27. Morrissey, D. V., Blanchard, K., Shaw, L., Jensen, K., Lockridge, J. A., Dickinson, B., McSwiggen, J. A., Vargeese, C., Bowman, K., Shaffer, C. S., Polisky, B. A., and Zinnen, S. (2005) Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication, Hepatology 41, 1349-1356. 28. Geisbert, T. W., Hensley, L. E., Kagan, E., Yu, E. Z., Geisbert, J. B., Daddario- DiCaprio, K., Fritz, E. A., Jahrling, P. B., McClintock, K., Phelps, J. R., Lee, A. C. H., Judge, A., Jeffs, L. B., and MacLachlan, I. (2006) Postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by RNA interference, J. Infect. Dis. 193, 1650-1657. 29. Dorn, G., Patel, S., Wotherspoon, G., Hemmings-Mieszczak, M., Barclay, J., Natt, F. J. C., Martin, P., Bevan, S., Fox, A., Ganju, P., Wishart, W., and Hall, J. (2004) siRNA relieves chronic neuropathic pain, Nucleic Acids Res. 32, e49-e49. 30. Kumar, P., Lee, S. K., Shankar, P., and Manjunath, N. (2006) A single siRNA suppresses fatal encephalitis induced by two different flaviviruses, PLoS Med. 3, e96. 31 31. Niu, X. Y., Peng, Z. L., Duan, W. Q., Wang, H., and Wang, P. (2006) Inhibition of HPV 16 E6 oncogene expression by RNA interference in vitro and in vivo, Int. J. Gynecol. Cancer 16, 743-751. 32. Takei, Y., Kadomatsu, K., Yuzawa, Y., Matsuo, S., and Muramatsu, T. (2004) A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics, Cancer Res. 64, 3365-3370. 33. Gurzov, E. N., and Izquierdo, M. (2005) RNA interference against Hec1 inhibits tumor growth in vivo, Gene Ther. 13, 1-7. 34. Pulukuri, S. M., Gondi, C. S., Lakka, S. S., Jutla, A., Estes, N., Gujrati, M., and Rao, J. S. (2005) RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo, J. Biol. Chem. 280, 36529-36540. 35. Takeshita, F., and Ochiya, T. (2006) Therapeutic potential of RNA interference against cancer, Cancer Sci. 97, 689-696. 36. Hu-Lieskovan, S., Heidel, J. D., Bartlett, D. W., Davis, M. E., and Triche, T. J. (2005) Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA Inhibits tumor growth in a murine model of metastatic Ewing's sarcoma, Cancer Res. 65, 8984-8992. 37. Bumcrot, D., Manoharan, M., Koteliansky, V., and Sah, D. W. (2006) RNAi therapeutics: a potential new class of pharmaceutical drugs, Nat. Chem. Biol. 2, 711-719. 38. Dorn, G., Patel, S., Wotherspoon, G., Hemmings-Mieszczak, M., Barclay, J., Natt, F. J. C., Martin, P., Bevan, S., Fox, A., Ganju, P., Wishart, W., and Hall, J. (2004) siRNA relieves chronic neuropathic pain, Nucleic Acids Res. 32, e49/41- e49/46. 39. Huang, L., and Liu, Y. (2011) In vivo delivery of RNAi with lipid-based nanoparticles, Annu. Rev. Biomed. Eng. 13, 507-530. 40. Kenny, G. D., Kamaly, N., Kalber, T. L., Brody, L. P., Sahuri, M., Shamsaei, E., Miller, A. D., and Bell, J. D. (2011) Novel multifunctional nanoparticle mediates siRNA tumour delivery, visualisation and therapeutic tumour reduction in vivo, J. Controlled Release 149, 111-116. 41. Wagner, E. (2011) Polymers for siRNA delivery: Inspired by viruses to be targeted, dynamic, and precise, Acc. Chem. Res. 45, 1005-1013. 32 42. Song, E., Zhu, P., Lee, S.-K., Chowdhury, D., Kussman, S., Dykxhoorn, D. M., Feng, Y., Palliser, D., Weiner, D. B., Shankar, P., Marasco, W. A., and Lieberman, J. (2005) Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors, Nat. Biotechnol. 23, 709-717. 43. McNamara, J. O., Andrechek, E. R., Wang, Y., Viles, K. D., Rempel, R. E., Gilboa, E., Sullenger, B. A., and Giangrande, P. H. (2006) Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras, Nat. Biotechnol. 24, 1005- 1015. 44. Dykxhoorn, D. M., Palliser, D., and Lieberman, J. (2006) The silent treatment: siRNAs as small molecule drugs, Gene Ther. 13, 541-552. 45. Shankar, P., Manjunath, N. N., and Lieberman, J. (2005) The prospect of silencing disease using RNA interference, J. Am. Med. Assoc. 293, 1367-1373. 46. Soutschek, J., Akinc, A., Bramlage, B., Charisse, K., Constien, R., Donoghue, M., Elbashir, S., Geick, A., Hadwiger, P., Harborth, J., John, M., Kesavan, V., Lavine, G., Pandey, R. K., Racie, T., Rajeev, K. G., Roehl, I., Toudjarska, I., Wang, G., Wuschko, S., Bumcrot, D., Koteliansky, V., Limmer, S., Manoharan, M., and Vornlocher, H.-P. (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs, Nature 432, 173-178. 47. Zimmermann, T. S., Lee, A. C., Akinc, A., Bramlage, B., Bumcrot, D., Fedoruk, M. N., Harborth, J., Heyes, J. A., Jeffs, L. B., John, M., Judge, A. D., Lam, K., McClintock, K., Nechev, L. V., Palmer, L. R., Racie, T., Rohl, I., Seiffert, S., Shanmugam, S., Sood, V., Soutschek, J., Toudjarska, I., Wheat, A. J., Yaworski, E., Zedalis, W., Koteliansky, V., Manoharan, M., Vornlocher, H. P., and MacLachlan, I. (2006) RNAi-mediated gene silencing in non-human primates, Nature 441, 111-114. 48. Pal, A., Ahmad, A., Khan, S., Sakabe, I., Zhang, C., Kasid, U. N., and Ahmad, I. (2005) Systemic delivery of Raf siRNA using cationic cardiolipin liposomes silences Raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer, Int. J. Oncol. 26, 1087-1091. 49. Schiffelers, R. M., Ansari, A., Xu, J., Zhou, Q., Tang, Q., Storm, G., Molema, G., Lu, P. Y., Scaria, P. V., and Woodle, M. C. (2004) Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle, Nucleic Acids Res. 32, e149-e149. 50. Takei, Y., Kadomatsu, K., Yuzawa, Y., Matsuo, S., and Muramatsu, T. (2004) A Small Interfering RNA Targeting Vascular Endothelial Growth Factor as Cancer Therapeutics, Cancer Res. 64, 3365-3370. 33 51. Watts, J. K., and Corey, D. R. (2010) Clinical status of duplex RNA, Bioorg. Med. Chem. Lett. 20, 3203-3207. 52. Melnikova, I. (2007) RNA-based therapies, Nat. Rev. Drug Discov. 6, 863-864. 53. Melnikova, I. (2005) Wet age-related macular degeneration, Nat. Rev. Drug. Discov. 4, 711-712. 54. Shoshani, T., Faerman, A., Mett, I., Zelin, E., Tenne, T., Gorodin, S., Moshel, Y., Elbaz, S., Budanov, A., Chajut, A., Kalinski, H., Kamer, I., Rozen, A., Mor, O., Keshet, E., Leshkowitz, D., Einat, P., Skaliter, R., and Feinstein, E. (2002) Identification of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis, Mol. Cell. Biol. 22, 2283-2293. 55. Nguyen, Q. D., Schachar, R. A., Nduaka, C. I., Sperling, M., Basile, A. S., Klamerus, K. J., Chi-Burris, K., Yan, E., Paggiarino, D. A., Rosenblatt, I., Khan, A., Aitchison, R., and Erlich, S. S. (2012) Phase 1 dose-escalation study of a siRNA targeting the RTP801 gene in age-related macular degeneration patients, Eye, 1-7. 56. Alvarez, R., Elbashir, S., Borland, T., Toudjarska, I., Hadwiger, P., John, M., Roehl, I., Morskaya, S. S., Martinello, R., Kahn, J., Van Ranst, M., Tripp, R. A., DeVincenzo, J. P., Pandey, R., Maier, M., Nechev, L., Manoharan, M., Kotelianski, V., and Meyers, R. (2009) RNA interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy, Antimicrob. Agents Ch. 53, 3952-3962. 57. Smith, F. J. D., Hickerson, R. P., Sayers, J. M., Reeves, R. E., Contag, C. H., Leake, D., Kaspar, R. L., and McLean, W. H. I. (2007) Development of therapeutic siRNAs for pachyonychia congenita, J. Invest. Dermatol. 128, 50-58. 58. Leachman, S. A., Hickerson, R. P., Schwartz, M. E., Bullough, E. E., Hutcherson, S. L., Boucher, K. M., Hansen, C. D., Eliason, M. J., Srivatsa, G. S., Kornbrust, D. J., Smith, F. J. D., McLean, W. H. I., Milstone, L. M., and Kaspar, R. L. (2009) First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder, Mol. Ther. 18, 442-446. 59. Shim, M. S., and Kwon, Y. J. (2010) Efficient and targeted delivery of siRNA in vivo, FEBS Journal 277, 4814-4827. 60. Kleinman, M. E., Yamada, K., Takeda, A., Chandrasekaran, V., Nozaki, M., Baffi, J. Z., Albuquerque, R. J. C., Yamasaki, S., Itaya, M., Pan, Y., Appukuttan, B., Gibbs, D., Yang, Z., Kariko, K., Ambati, B. K., Wilgus, T. A., DiPietro, L. A., Sakurai, E., Zhang, K., Smith, J. R., Taylor, E. W., and Ambati, J. (2008) Sequence- and target-independent angiogenesis suppression by siRNA via TLR3, Nature 452, 591-597. 34 61. Duncan, R. (2011) Polymer therapeutics as nanomedicines: new perspectives, Curr. Opin. Biotechnol. 22, 492-501. 62. Meng, F., Hennink, W. E., and Zhong, Z. (2009) Reduction-sensitive polymers and bioconjugates for biomedical applications, Biomaterials 30, 2180-2198. 63. Smith, M. H., and Lyon, L. A. (2011) Multifunctional nanogels for siRNA Delivery, Acc. Chem. Res. 45, 985-993. 64. Son, S., Namgung, R., Kim, J., Singha, K., and Kim, W. J. (2011) Bioreducible polymers for gene silencing and delivery, Acc. Chem. Res. 45, 1100-1112. 65. Itaka, K., Kanayama, N., Nishiyama, N., Jang, W.-D., Yamasaki, Y., Nakamura, K., Kawaguchi, H., and Kataoka, K. (2004) Supramolecular nanocarrier of siRNA from PEG-based block catiomer carrying diamine side chain with distinctive pKa directed to enhance intracellular gene silencing, J. Am. Chem. Soc. 126, 13612- 13613. 66. Oishi, M., Nagasaki, Y., Itaka, K., Nishiyama, N., and Kataoka, K. (2005) Lactosylated poly(ethylene glycol)-siRNA conjugate through acid-labile β- thiopropionate Linkage to construct pH-sensitive polyion complex micelles achieving enhanced gene silencing in hepatoma cells, J. Am. Chem. Soc. 127, 1624-1625. 67. Rudzinski, W. E., and Aminabhavi, T. M. (2010) Chitosan as a carrier for targeted delivery of small interfering RNA, Int. J. Pharm. 399, 1-11. 68. Meyer, M., Philipp, A., Oskuee, R., Schmidt, C., and Wagner, E. (2008) Breathing life into polycations: functionalization with pH-Responsive endosomolytic peptides and polyethylene glycol enables siRNA delivery, J. Am. Chem. Soc. 130, 3272-3273. 69. Ornelas-Megiatto, C., Wich, P. R., and Frechet, J. M. (2012) Polyphosphonium polymers for siRNA delivery: An efficient and nontoxic alternative to polyammonium carriers, J. Am. Chem. Soc. 134, 1902-1905. 70. Krebs, M. D., and Alsberg, E. (2011) Localized, targeted, and sustained siRNA delivery, Chem. Eur. J. 17, 3054-3062. 71. Yezhelyev, M. V., Qi, L., O'Regan, R. M., Nie, S., and Gao, X. (2008) Proton-sponge coated quantum dots for siRNA delivery and intracellular imaging, J. Am. Chem. Soc. 130, 9006-9012. 72. Qi, L., and Gao, X. (2008) Quantum dot−amphipol nanocomplex for intracellular delivery and real-time imaging of siRNA, ACS Nano 2, 1403-1410. 35 73. Li, W., and Szoka, F. C., Jr. (2007) Lipid-based nanoparticles for nucleic acid delivery, Pharm. Res. 24, 438-449. 74. Akinc, A., Zumbuehl, A., Goldberg, M., Leshchiner, E. S., Busini, V., Hossain, N., Bacallado, S. A., Nguyen, D. N., Fuller, J., Alvarez, R., Borodovsky, A., Borland, T., Constien, R., de Fougerolles, A., Dorkin, J. R., Narayanannair Jayaprakash, K., Jayaraman, M., John, M., Koteliansky, V., Manoharan, M., Nechev, L., Qin, J., Racie, T., Raitcheva, D., Rajeev, K. G., Sah, D. W., Soutschek, J., Toudjarska, I., Vornlocher, H. P., Zimmermann, T. S., Langer, R., and Anderson, D. G. (2008) A combinatorial library of lipid-like materials for delivery of RNAi therapeutics, Nat. Biotechnol. 26, 561-569. 75. Whitehead, K. A., Langer, R., and Anderson, D. G. (2009) Knocking down barriers: advances in siRNA delivery, Nat. Rev. Drug. Discov. 8, 129-138. 76. Manoharan, M., Elbashir, S., and Harborth, J. (2004) SiRNA and cholesterol-siRNA conjugates for reducing apoB-100, glucose-6-phosphatase, and β-catenin levels and treatment of disease, US Patent 2004/091515. 77. Wolfrum, C., Shi, S., Jayaprakash, K. N., Jayaraman, M., Wang, G., Pandey, R. K., Rajeev, K. G., Nakayama, T., Charrise, K., Ndungo, E. M., Zimmermann, T., Koteliansky, V., Manoharan, M., and Stoffel, M. (2007) Mechanisms and optimization of in vivo delivery of lipophilic siRNAs, Nat. Biotechnol. 25, 1149- 1157. 78. Kam, N. W. S., Liu, Z., and Dai, H. (2005) Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing, J. Am. Chem. Soc. 127, 12492-12493. 79. Bartlett, D. W., Su, H., Hildebrandt, I. J., Weber, W. A., and Davis, M. E. (2007) Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging, Proc. Natl. Acad. Sci. USA 104, 15549-15554. 80. Nayak, S., Lee, H., Chmielewski, J., and Lyon, L. A. (2004) Folate-mediated cell targeting and cytotoxicity using thermoresponsive microgels, J. Am. Chem. Soc. 126, 10258-10259. 81. Guo, S., Huang, F., and Guo, P. (2006) Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells, Gene Ther. 13, 814-820. 82. Zhu, L., and Mahato, R. I. (2010) Targeted delivery of siRNA to hepatocytes and hepatic stellate cells by bioconjugation, Bioconjugate Chem. 21, 2119-2127. 36 83. Crombez, L., Aldrian-Herrada, G., Konate, K., Nguyen, Q. N., McMaster, G. K., Brasseur, R., Heitz, F., and Divita, G. (2008) A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells, Mol. Ther. 17, 95-103. 84. Kumar, P., Wu, H., McBride, J. L., Jung, K.-E., Hee Kim, M., Davidson, B. L., Kyung Lee, S., Shankar, P., and Manjunath, N. (2007) Transvascular delivery of small interfering RNA to the central nervous system, Nature 448, 39-43. 85. Zhou, J., Li, H., Li, S., Zaia, J., and Rossi, J. J. (2008) Novel dual inhibitory function aptamer-siRNA delivery system for HIV-1 therapy, Mol. Ther. 16, 1481- 1489. 86. Zhou, J., Swiderski, P., Li, H., Zhang, J., Neff, C. P., Akkina, R., and Rossi, J. J. (2009) Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells, Nucleic Acids Res. 37, 3094-3109. 87. Chu, T. C., Twu, K. Y., Ellington, A. D., and Levy, M. (2006) Aptamer mediated siRNA delivery, Nucleic Acids Res. 34, e73-e73. 88. Braasch, D. A., Jensen, S., Liu, Y., Kaur, K., Arar, K., White, M. A., and Corey, D. R. (2003) RNA interference in mammalian cells by chemically-modified RNA, Biochemistry 42, 7967-7975. 89. Chiu, Y.-L., and Rana, T. M. (2003) SiRNA function in RNAi: A chemical modification analysis, RNA 9, 1034-1048. 90. Allerson, C. R., Sioufi, N., Jarres, R., Prakash, T. P., Naik, N., Berdeja, A., Wanders, L., Griffey, R. H., Swayze, E. E., and Bhat, B. (2005) Fully 2‘-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA, J. Med. Chem. 48, 901-904. 91. Egli, M., Minasov, G., Tereshko, V., Pallan, P. S., Teplova, M., Inamati, G. B., Lesnik, E. A., Owens, S. R., Ross, B. S., Prakash, T. P., and Manoharan, M. (2005) Probing the influence of stereoelectronic effects on the biophysical properties of oligonucleotides: comprehensive analysis of the RNA affinity, nuclease resistance, and crystal structure of ten 2‘-O-ribonucleic acid modifications, Biochemistry 44, 9045-9057. 92. Prakash, T. P., Allerson, C. R., Dande, P., Vickers, T. A., Sioufi, N., Jarres, R., Baker, B. F., Swayze, E. E., Griffey, R. H., and Bhat, B. (2005) Positional effect of chemical modifications on short interference RNA activity in mammalian cells, J. Med. Chem. 48, 4247-4253. 37 93. Hoerter, J. A., and Walter, N. G. (2007) Chemical modification resolves the asymmetry of siRNA strand degradation in human blood serum, RNA 13, 1887- 1893. 94. Parrish, S., Fleenor, J., Xu, S., Mello, C., and Fire, A. (2000) Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference, Mol. Cell 6, 1077-1087. 95. Dowler, T., Bergeron, D., Tedeschi, A.-L., Paquet, L., Ferrari, N., and Damha, M. J. (2006) Improvements in siRNA properties mediated by 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (FANA), Nucleic Acids Res. 34, 1669-1675. 96. Manoharan, M., Akinc, A., Pandey, R. K., Qin, J., Hadwiger, P., John, M., Mills, K., Charisse, K., Maier, M. A., Nechev, L., Greene, E. M., Pallan, P. S., Rozners, E., Rajeev, K. G., and Egli, M. (2011) Unique gene-silencing and structural properties of 2′-fluoro-modified siRNAs, Angew. Chem. Int. Ed. 50, 2284-2288. 97. Fauster, K., Hartl, M., Santner, T., Aigner, M., Kreutz, C., Bister, K., Ennifar, E., and Micura, R. (2012) 2′-Azido RNA, a versatile tool for chemical biology: Synthesis, X-ray structure, siRNA applications, click labeling, ACS Chem. Biol. 7, 581-589. 98. Watts, J. K., Choubdar, N., Sadalapure, K., Robert, F., Wahba, A. S., Pelletier, J., Pinto, B. M., and Damha, M. J. (2007) 2'-Fluoro-4'-thioarabino-modified oligonucleotides: conformational switches linked to siRNA activity, Nucleic Acids Res. 35, 1441-1451. 99. Gore, K. R., Nawale, G. N., Harikrishna, S., Chittoor, V. G., Pandey, S. K., Höbartner, C., Patankar, S., and Pradeepkumar, P. I. (2012) Synthesis, gene silencing, and molecular modeling studies of 4′-C-aminomethyl-2′-O-methyl modified small interfering RNAs, J. Org. Chem. 77, 3233-3245. 100. Manoharan, M. (2004) RNA interference and chemically modified small interfering RNAs, Curr. Opin. Chem. Biol. 8, 570-579. 101. Rozners, E. (2006) Carbohydrate chemistry for RNA interference: synthesis and properties of RNA analogues modified in sugar-phosphate backbone, Curr. Org. Chem. 10, 675-692. 102. Watts, J. K., Deleavey, G. F., and Damha, M. J. (2008) Chemically modified siRNA: tools and applications, Drug Discov. Today 13, 842-855. 103. Manoharan, M., and Rajeev, K. G. (2009) Non-natural ribonucleosides with modified ribose and base moieties for use use in siRNAs and their synthesis, US Patent 2009/120878. 38 104. Deleavey, G. F., Watts, J. K., Alain, T., Robert, F., Kalota, A., Aishwarya, V., Pelletier, J., Gewirtz, A. M., Sonenberg, N., and Damha, M. J. (2010) Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing, Nucleic Acids Res. 38, 4547-4557. 105. Corey, D. R. (2007) Chemical modification: the key to clinical application of RNA interference?, The Journal of clinical investigation 117, 3615-3622. 106. Bramsen, J. B., Pakula, M. M., Hansen, T. B., Bus, C., Langkjaer, N., Odadzic, D., Smicius, R., Wengel, S. L., Chattopadhyaya, J., Engels, J. W., Herdewijn, P., Wengel, J., and Kjems, J. (2010) A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects, Nucleic Acids Res. 38, 5761-5773. 107. Elbashir, S. M., Martinez, J., Patkaniowska, A., Lendeckel, W., and Tuschl, T. (2001) Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate, EMBO J. 20, 6877-6888. 108. Czauderna, F., Fechtner, M., Dames, S., Aygün, H., Klippel, A., Pronk, G. J., Giese, K., and Kaufmann, J. (2003) Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells, Nucleic Acids Res. 31, 2705-2716. 109. Prakash, T. P., Allerson, C. R., Dande, P., Vickers, T., Sioufi, N., Jarres, R., Baker, B., Swayze, E. E., Griffey, R. H., and Bhat, B. (2004) Positional effects of chemical modification on siRNA activity, In Abstracts of Papers, 227th ACS National Meeting, Anaheim, CA. , pp MEDI-175, American Chemical Society. 110. Hoshika, S., Minakawa, N., and Matsuda, A. (2005) RNA interference induced by siRNAs modified with 4'-thioribonucleosides, Nucleic Acids Symp. Ser. 49, 77- 78. 111. Hoshika, S., Minakawa, N., Kamiya, H., Harashima, H., and Matsuda, A. (2005) RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured mammalian cells, FEBS Lett. 579, 3115-3118. 112. Nauwelaerts, K., Fisher, M., Froeyen, M., Lescrinier, E., Van Aerschot, A., Xu, D., DeLong, R., Kang, H., Juliano, R. L., and Herdewijn, P. (2007) Structural characterization and biological evaluation of small interfering RNAs containing cyclohexenyl nucleosides, J. Am. Chem. Soc. 129, 9340-9348. 113. Harborth, J., Elbashir, S. M., Vandenburgh, K., Manninga, H., Scaringe, S. A., Weber, K., and Tuschl, T. (2003) Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing, Antisense Nucleic Acid Drug Dev. 13, 83-105. 39 114. Geary, R. S., Yu, R. Z., and Levin, A. A. (2001) Pharmacokinetics of phosphorothioate antisense oligodeoxynucleotides, Curr. Opin. Invest. Drugs 2, 562-573. 115. Hall, A. H. S., Wan, J., Shaughnessy, E. E., Ramsay, S. B., and Alexander, K. A. (2004) RNA interference using boranophosphate siRNAs: structure-activity relationships, Nucleic Acids Res. 32, 5991-6000. 116. Li, P., Sergueeva, Z. A., Dobrikov, M., and Shaw, B. R. (2007) Nucleoside and oligonucleoside boranophosphates: chemistry and properties, Chem. Rev. 107, 4746-4796. 117. Potenza, N., Moggio, L., Milano, G., Salvatore, V., Di, B. B., Russo, A., and Messere, A. (2008) RNA interference in mammalia cells by RNA-3'-PNA chimeras, Int. J. Mol. Sci. 9, 299-315. 118. Zhang, N., Tan, C., Cai, P., Zhang, P., Zhao, Y., and Jiang, Y. (2009) RNA interference in mammalian cells by siRNAs modified with morpholino nucleoside analogues, Bioorg. Med. Chem. 17, 2441-2446. 119. Efthymiou, T. C., Huynh, V., Oentoro, J., Peel, B., and Desaulniers, J.-P. (2012) Efficient synthesis and cell-based silencing activity of siRNAS that contain triazole backbone linkages, Bioorg. Med. Chem. Lett. 22, 1722-1726. 120. Meyer, M., Dohmen, C., Philipp, A., Kiener, D., Maiwald, G., Scheu, C., Ogris, M., and Wagner, E. (2009) Synthesis and biological evaluation of a bioresponsive and endosomolytic siRNA−polymer conjugate, Mol. Pharm. 6, 752-762. 121. Grijalvo, S., Ocampo, S. M., Perales, J. C., and Eritja, R. (2010) Synthesis of oligonucleotides carrying amino lipid groups at the 3'-end for RNA interference studies, J. Org. Chem. 75, 6806-6813. 122. Yamada, T., Peng, C. G., Matsuda, S., Addepalli, H., Jayaprakash, K. N., Alam, M. R., Mills, K., Maier, M. A., Charisse, K., Sekine, M., Manoharan, M., and Rajeev, K. G. (2011) Versatile site-specific conjugation of small molecules to siRNA using click chemistry, J. Org. Chem. 76, 1198-1211. 123. Cesarone, G., Edupuganti, O. P., Chen, C.-P., and Wickstrom, E. (2007) Insulin receptor substrate 1 knockdown in human MCF7 ER+ breast cancer cells by nuclease-resistant IRS1 siRNA conjugated to a disulfide-bridged d-peptide analogue of insulin-like growth factor 1, Bioconjugate Chem. 18, 1831-1840. 124. De Paula, D., Bentley, M. V., and Mahato, R. I. (2007) Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting, RNA 13, 431-456. 40 125. Puthenveetil, S., Whitby, L., Ren, J., Kelnar, K., Krebs, J. F., and Beal, P. A. (2006) Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification, Nucleic Acids Res. 34, 4900-4911. 126. Somoza, A., Chelliserrykattil, J., and Kool, E. T. (2006) The roles of hydrogen bonding and sterics in RNA interference, Angew. Chem. Int. Ed. 45, 4994-4997. 127. Xia, J., Noronha, A., Toudjarska, I., Li, F., Akinc, A., Braich, R., Frank- Kamenetsky, M., Rajeev, K. G., Egli, M., and Manoharan, M. (2006) Gene silencing activity of siRNAs with a ribo-difluorotoluyl nucleotide, ACS Chem. Biol. 1, 176-183. 128. Terrazas, M., and Kool, E. T. (2009) RNA major groove modifications improve siRNA stability and biological activity, Nucleic Acids Res. 37, 346-353. 129. Peacock, H., Fostvedt, E., and Beal, P. A. (2010) Minor-groove-modulating adenosine replacements control protein binding and RNAi activity in siRNAs, ACS Chem. Biol. 5, 1115-1124. 130. Kannan, A., Fostvedt, E., Beal, P. A., and Burrows, C. J. (2011) 8-Oxoguanosine switches modulate the activity of alkylated siRNAs by controlling steric effects in the major versus minor grooves, J. Am. Chem. Soc. 133, 6343-6351. 131. Wahba, A. S., Azizi, F., Deleavey, G. F., Brown, C., Robert, F., Carrier, M., Kalota, A., Gewirtz, A. M., Pelletier, J., Hudson, R. H. E., and Damha, M. J. (2011) Phenylpyrrolocytosine as an unobtrusive base modification for monitoring activity and cellular trafficking of siRNA, ACS Chem. Biol. 6, 912-919. 132. Lima, W. F., Wu, H., Nichols, J. G., Sun, H., Murray, H. M., and Crooke, S. T. (2009) Binding and cleavage specificities of human argonaute2, J. Biol. Chem. 284, 26017-26028. 133. Somoza, A., Silverman, A. P., Miller, R. M., Chelliserrykattil, J., and Kool, E. T. (2008) Steric Effects in RNA Interference: Probing the Influence of Nucleobase Size and Shape, Chem. Eur. J. 14, 7978-7987. 134. Addepalli, H., Meena, Peng, C. G., et al. (2010) Modulation of thermal stability can enhance the potency of siRNA, Nucleic Acids Res. 38, 7320-7331. 135. Sipa, K., Sochacka, E., Kazmierczak-Baranska, J., Maszewska, M., Janicka, M., Nowak, G., and Nawrot, B. (2007) Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA, RNA 13, 1301-1316. 136. Mikat, V., and Heckel, A. (2007) Light-dependent RNA interference with nucleobase-caged siRNAs, RNA 13, 2341-2347. CHAPTER 2 8-ALKOXYADENOSINES AS POTENTIAL MODULATORS OF OFF-PATHWAY PROTEIN BINDING Introduction SiRNA-based RNAi pharmaceuticals are gradually becoming a principal alternative to small molecule-based drugs. Theoretically, any malfunctioning gene (and the related disease) can be targeted by this technique; even so called ‘nondruggable' targets can be reached by siRNAs. RNAi is still a relatively new technology, and within a short time period significant advancement has been made to bring this technique from the research laboratory to the clinic. However, the therapeutic potential of siRNA has not yet been maximized mainly due to delivery problems and undesired side effects, such as sequence-dependent off-target effects, sequence-specific and sequence-independent off-pathway protein binding leading to innate immune responses. Although not as pronounced as the long dsRNAs, siRNAs can induce an innate immune response. SiRNAs are recognized by the intracellular defense mechanism as a potential viral infection; and the genes related to the innate immune system (such as INFα, 2´,5´-oligoadenylate synthase (OAS) and signal transducers/activators of transcription) are upregulated leading to immunostimulation (1, 2). These side effects can mask or even counter RNAi efficacy and specificity. Due to these off-target effects and off-pathway protein binding, the therapeutic dosage of the 42 siRNA must be increased significantly leading to dsRNA-related toxicity. Hence, prevention of these undesired side effects is essential. Off-target gene silencing. Initially, siRNA was thought to be an extremely selective method of gene silencing; it was thought to be only functional if there was a perfect complementarity between the guide strand and the mRNA. Several studies have claimed that even a single mismatch with the mRNA completely abolished the RNAi activity (4). However, it was gradually understood that siRNA could silence genes, partially or extensively, even when there is partial complementarity between the siRNA and the mRNA. Jackson et al. reported that this kind of miRNA-like effects could originate when any region of an mRNA is complementary to the seed region (bases at positions 2-8) of the guide siRNA (5-7). One probable solution to this ‘off-target' effect is to select a different siRNA, the seed region of which is only complementary to the target mRNA. Sometimes this might not be trivial because many other parameters should also be satisfied to design a successful siRNA. Another alternative is to chemically modify specific positions of the seed region to reduce the off-target effects. A 2´-OMe modification in the seed region significantly lowered the sequence-dependent off-target effects. This modification, when placed in position 2, is more effective than any other positions (5). Bramsen et al. reported several ribose modifications that are effective in preventing the sequence-dependent off-target effect; their study revealed that the UNA modification is the most effective modification out of ten different modifications (Figure 2.1) (8). The UNA modification weakens the siRNA-mRNA interaction moderately, without lowering the RNAi efficacy considerably. 43 Figure 2.1. Ribose modifications to prevent off-target gene silencing (8). O O OH O Base UNA O O O O Base 2'-methoxy O O O O Base LNA O O O O Base NH3 2'-EA O O O Base O O O Base OH ANA HNA O O O Base HO HM CLNA CENA AENA O O O Base O O O Base O O NH O Base 44 Sequence-specific immunostimulation. Analogous to 5´-CpG-3´ containing DNA oligonucleotides (9), certain siRNA sequences are also immunostimulatory. GU-rich siRNA sequences produces Interferon-α (INFα) which upregulates INFα-associated genes in human peripheral blood mononuclear cells (PBMCs), plasmacytoid dendritic cells (PDCs) isolated from human peripheral blood, as well as in CD4+ 4 and CD8+ T cells in mice (10, 11). In these cases, immunostimulation occurs through interaction of Toll-like receptor 7 (TLR7) with certain sequence motifs in the guide or the passenger strand. The mechanism of TLR-mediated immunostimulation due to siRNA binding is currently unknown. Several ribose modifications such as 2´-OMe, LNA, 2´-F-RNA, 2´-F-ANA, are known to prevent TLR-mediated immunostimulation (Figure 2.2) (10, 12, 13). To date, no base modification has been reported to address this off-pathway interaction. The TLR7-mediated innate immune response is sequence-dependent, so recognition of specific nucleoside motifs (such as GU-rich siRNAs) might be prevented by appropriate base modifications. Sequence-independent off-pathway protein binding and immunostimulation. SiRNAs, irrespective of their sequences, can bind with proteins that are not directly involved in the RNAi pathway (1, 14). Among these proteins, an important class contains double-stranded RNA binding motif (dsRBM) containing domains, which can be used to bind duplex RNAs longer than 16 base pairs. Some important members of this group of proteins are RNA-dependent protein kinase (PKR) (Figure 2.3) and adenosine deaminases that modify RNA (ADARs), RNA helicase A, and Staufen1, an RNA trafficking protein. Duplex RNAs are also ligands of TLR3 and OAS and naturally can also interact with siRNA (2, 15). TLRs are a group of proteins that recognize molecular 45 Figure 2.2. Ribose modifications to prevent sequence-specific immunostimulation (13). O O F O Base 2'-F-RNA O O O O Base LNA O O O Base F 2'-FANA O O O O Base 2'-F-RNA 46 Figure 2.3. Structure and mechanism of activation of the RNA-dependent protein kinase (PKR). (a) and (b) structure of the PKR; (c) mechanism of activation of the monomeric PKR; (d) activation of PKR through dimerization (reproduced with permission from reference (3) ). 47 scaffolds related to pathogenic infection and initiate immune response (15). OAS can be stimulated upon binding with siRNA and nonspecific Ribonuclease L is activated as a part of the innate immune response to viral infection (16). PKR and ADAR1 have been reported to bind with siRNAs complicating RNAi experiments (1, 14). ADAR1 binds with the siRNA and reduces its effective concentration in cell, affecting RNAi efficacy significantly (14). PKR is a crucial component for controlling translation initiation and signal transduction; upon binding with siRNA, PKR sends signals analogous to viral infection and instigates interferon-mediated immunostimulatory response, which finally leads to cell apoptosis (Figure 2.3). Activation of PKR also induces global expression of PKR-associated genes in the human glioblastoma cell line (1). Nucleobase modifications to prevent the siRNA-PKR interaction. Prevention of these sequence-independent off-pathway protein interactions is essential for the potency and specificity of therapeutic siRNA. The Beal laboratory initiated a systematic study on base modifications of the siRNA to address and alleviate siRNA-PKR interaction (17, 18). During PKR-binding studies with stem-loop IV of EBER1 (an RNA from Epstein- Barr virus), they noticed that substitution of some specific guanosines with N2- benzylguanosine could greatly diminish PKR-EBER1 interaction. It was also realized that this strategy of ‘steric occlusion of the minor groove' could be utilized to address siRNA off-target effects (Figure 2.4). Their hypothesis was experimentally verified when modifications at several positions (6, 9, 11, 14) of the sense strand of caspase 2 siRNA successfully prevented PKR binding (Figure 2.4) (18). These N2-benzylguanosine analogs maintained normal 48 Figure 2.4. Minor groove purine modifications to prevent PKR binding (18-20). NH N N N O NH2 HN N O O G6 U33 NH N N N O NH HN N O O BnG6 U33 minor groove major groove 5' - GGAAAUGCAAGAGAAACUGdTdT - 3' passenger strand 3' - dTdTCCUUUACGUUCUCUUUGAC - 5' guide strand Caspase 2 siRNA N2-benzylation of G in the G:U wobble pair in stem-loop RNA of EBV NH N N N O NH N N N O N2-benzylG C H H GG GUCUG GGC GGG - 5' CC-CAGAC CUG-CUC - 3' U U A U G C UU U UU 10 20 30 Stem-loop RNA of EBV N N N N NH HN N O O N2-alkyl-2-aminopurine U R major groove major groove minor groove minor groove prevented PKR binding N N N N N N N R H O O OH OH NH OH HO 49 hydrogen-bonded base pairing while projecting sterically demanding moieties in the minor groove; thereby preventing binding of dsRBM (of PKR) onto minor grooves of siRNA duplexes (Figure 2.5). Peacock et al. have shown (Figure 2.4) that pendent minor groove modifications of 2-aminopurines (20) in the passenger caspase 2 siRNA can also successfully prevent siRNA-PKR interaction, while maintaining RNAi efficacy. Similar modifications of the minor or major groove in the guide strand might serve multiple purposes, such as preventing PKR or ADAR1 binding and elucidating novel mechanistic features of siRNA-RISC interactions while maintaining RNA interference activity. However, siRNA base modifications in the guide strand are more challenging; Some base modifications drastically reduced or completely abolished the RNAi efficacy when placed at crucial siRNA sites, such as in the seed region or at the cleavage site. N2-Alkylated 2´-deoxy-7,8-dihydro-8-oxoguanosine containing siRNAs could prevent siRNA-PKR interaction significantly by placing a ‘switchable' alkyl group in the minor groove of an siRNA during delivery; in the RISC, the modified siRNAs can flip its steric bulk into the major groove, and maintain good siRNA efficacy (Figure 2.6) (21). However, oxidized purine lesions, such as 2´-deoxy-7,8-dihydro-8-oxoguanosine, are known to be immunostimulatory themselves. Therefore exploring other purine modifications in the guide strand is worthwhile and essential. Here, we report a series of novel nucleobase modifications, 8-alkoxyAs and their potential application in the RNAi. 8-AlkoxyA phosphoramidites were synthesized and incorporated into the guide strand of a caspase 2 siRNA through solid phase oligonucleotide synthesis. Modified siRNAs were tested for their RNAi efficacy and ability to address off-target effects due to siRNA-PKR interactions. 50 Figure 2.5. Sites of the siRNA passenger strand modifications to prevent PKR binding. (reproduced with permission from reference (18)). 51 Figure 2.6. ‘Switchable' N2-alkylated 2´-deoxy-7,8-dihydro-8-oxoguanosines in duplex RNA to prevent PKR binding: (A) cartoon (B) base-pairs. (adapted and reproduced with permission from reference (21). A) B) 52 8-Substituted adenosines and guanosines have been shown to exist in an equilibrium mixture of syn/anti conformations. Accordingly, 8-alkoxyadenosines are postulated to have a tendency to flip between anti and syn conformations, depending on the base-pairing partner. In the natural anti conformation, 8-alkoxyA will base pair with U, whereas, in the syn conformation, the Hoogsteen face of the nucleoside will be exposed for base pairing and it will complement best with anti G (Figure 2.7). We propose that during delivery of the siRNA, 8-alkoxyA in the guide strand (opposite to G in the passenger strand) would project its steric blockade into the minor groove of siRNA, thereby preventing intracellular protein binding onto the RNA. When the siRNA is recruited into the RISC assembly, the 8-alkoxyA in the guide siRNA would encounter U in the mRNA and would flip its steric bulk into the major groove, thereby allowing necessary guide strand-mRNA-RISC assembly to form (Figure 2.8). Alkyl groups were chosen based on their size and shape: propargyl, phenethyl and cyclohexylethyl. The rationale behind the choices is that: smaller groups (propargyl) might exhibit higher duplex stabilities and better mRNA knock down efficiencies whereas larger group (cyclohexylethyl) might prevent immunostimulation to a greater extent, and a medium-sized group (such as phenethyl) might serve both the purposes equally efficiently. Synthesis of the 8-alkoxyadenosine phosphoramidites. 8-Alkoxyadenosine phosphoramidites were synthesized in multiple steps from a ribose-protected 8- bromoadenosine derivative. Here it is outlined briefly, and the detailed step- by-step synthesis can be found in the Experimental section below. Adenosine was first brominated at C8 following a published procedure and the hydroxyl groups of the ribose 53 Figure 2.7. Proposed base-pairing of the 8-alkoxyadenosines. N N N N N O N O O N H H H N N N N N O H H R N N N O N N H H H R 8-alkoxyAsyn : Ganti 8-alkoxyAanti : Uanti R 54 Figure 2.8. Proposed base ‘switch' cartoon to prevent off-pathway protein interaction while maintaining RNAi efficacy. Passenger U Guide G minor groove mRNA RISC major groove Guide steric blockade hidden in the major groove for protein interactions in the RISC steric blockade in the minor groove to prevent off-pathway protein binding A A 55 moiety were protected by silylating agents. 5´-OH and 3´-OH groups were protected using a bidentate silylating agent and the remaining 2´-OH was protected using a TBDMS protecting group. Next, the bromine was displaced with an alkoxy groups that was generated in situ by adding dropwise n-BuLi into the corresponding alcohol in THF. The exocyclic amine of adenosine was protected using a benzoyl group. Then, the 5´-OH and 3´-OH were deprotected using pyridine-HF reagent, leaving the 2´-O-protection intact. For application in the automated solid phase oligonucleotide synthesis, the 5´-OH was protected with a DMT group, and the 3´-OH was coupled to a phosphoramidite group (Figure 2.9). Experimental All the chemicals were obtained commercially, unless otherwise stated, and used without further purification. Freshly distilled solvents were used where anhydrous solvents were required. THF was distilled from Na metal and benzophenone. For TLC, Merck silica gel 60 F254 pre-coated plates were used. Glassware for all the reactions was dried overnight in the oven at 150 °C and cooled in desiccators. Silica gel 60 (230-400 mesh) was used for flash chromatographic purpose. Column fractions were carried out as mentioned in the separation of individual compounds. 1H, 13C, and 31P NMR spectra were recorded at 300, 75, and 121 MHz, respectively. Chemical shift values are reported in parts per million (ppm) using the solvent peak as the reference. In interpreting the 1H NMR peak multiplicities, s, d, dd, t, q, m, and brs abbreviations were used for singlet, doublet, doublet of doublets, triplet, quartet, multiplet, and broad singlet, respectively. 56 Figure 2.9. Synthesis of the 8-alkoxyA phosphoramidites. N N N N NH2 O OH OH HO N N N N NH2 O OH OH HO NaOAc buffer pH 4 4 eqv Br2 / H2O 23 oC 4-5 h H Br (t-Bu)2Si(OTf)2 DMF, Imidazole, 0 oC, 1h N N N N NH2 O O OH O Br Si N N N N NH2 O O O O Br Si Si TBDMS-Cl DMF, Imidazole, 60 oC, 12 h 82.5% yield 98% yield over 2 steps 1 2 3 N N N N NH2 O O O O O Si Si i) PhCOCl Pyridine, 0-23 oC 89 - 95% 80 - 92% 84 - 92% ii) NH3/MeOH, 23 oC, 2 h R propargyl (Pg) phenethyl (Pe) cyclohexylethyl (Ce) 94% 97% 65% 5 4 10 9 15 14 N N N N HN O O O O O Si Si R O Ph n-BuLi / anh. THF, 23 oC, 24 h R OH 3 eqv. HF-pyridine, -10 oC, 2-2.5 h 82 - 95% 88 - 94% 87 - 90% 6 11 16 N N N N HN O OH O HO O Si R O Ph pyridine, CH2Cl2 R DMT-Cl, pyridine 0 oC, 12 h 75 - 80% 68 - 81% 74 - 78% 7 12 17 N N N N HN O OH O DMTO O Si R O Ph P Cl N O N DIPEA, anh. THF 23 oC, 6h N N N N HN O O O DMTO O Si R O Ph P N O N 82 - 90% 82 - 86% 78 - 85% 8 13 18 57 8-Bromoadenosine (1). 8-Bromoadenosine was synthesized following a literature procedure (22). Product yield was 82.5%: melting point >200 °C; 1H NMR (300 MHz, d6-DMSO) δ 8.12 (s, 1H, 2-H), 7.56 (s, 2 H, 6-NH2), 5.80-5.84 (d, 1H, 1´-H), 5.42-5.52 (m, 2H, 2´- and 3´-OH), 5.20-5.25 (d, 1H, 5´-OH), 5.03-5.12 (q, 1H, 2´-H), 4.17-4.22 (brs, 1H, 3´-H), 3.94-4.00 (m, 1H, 4´-H), 3.62-3.72 (m, 1H, 5´-H), 3.46-3.56 (m, 1H, 5´- H). 13C NMR (75 MHz, d6-DMSO): δ 155.2, 152.4, 149.8, 127.2, 119.6, 90.4, 86.7, 71.1, 70.9. HRMS: calcd for C10H12N5O4Na79Br [MNa+] 367.9970, obsd 367.9973. 5´,3´-O-Bis(-t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-8-bromoadenosine(3). Both the 3´-OH and 5´-OH were protected by a designer protecting group introduced by Trost and coworkers (23). In an oven-dried flask 1 mmol (346 mg) of 1 was suspended in 5 mL anhydrous DMF. The suspension was cooled down to 0 °C and 1.1 equivalent (400 μL) di-t-butylsilyl ditriflate was added drop wise under stirring condition, at the same temperature. The reaction was carried out under N2 atmosphere for 30 min and after that time period no starting material was detected, when the reaction mixture was analyzed using TLC. After synthesis of the intermediate 2, the reaction was quenched immediately with 5 equivalents (344 mg) of imidazole at 0 °C. The reaction was stirred at the same temperature for 5 additional min, and then the system was allowed to equilibrate to room temperature. Then, 1.2 equivalents (181 mg) of t-butyldimethylsilyl chloride were added and a reflux condenser was connected to the reaction flask. The temperature was elevated to 60 °C, and the reaction was run overnight under nitrogen. The suspension was cooled down to room temperature, water was added and the precipitate was collected by suction filtration. The supernatant was discarded, and then the white precipitate was rewashed with cold methanol (4 °C) to obtain pure compound 3. The methanol layer was 58 evaporated under reduced pressure and a simple column separation (5:1 hexane:ethyl acetate) could separate the remaining product. Overall yield of the product was 590 mg (98%): silica gel TLC Rf 0.44 (3:2 hexane:ethyl acetate); 1H NMR (300 MHz, CDCl3): δ 8.25 (s, 1H, 2-H), 6.55 (s, 2 H, 6-NH2), 5.89-5.92 (d, 1H, 1´-H), 5.15-5.24 (q, 1H, 2´-H), 4.86-4.92 (d, 1H, 3´-H), 4.33-4.42 (dd, 1H, 4´-H), 3.95-4.14 (m, 2H, 5´-H), 1.16 (s, 9H, (CH3)3), 1.03 (s, 9H, (CH3)3), 0.88 (s, 9H, (CH3)3), 0.11 (s, 3H, CH3), 0.07 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3): δ 154.9, 153.3, 150.7, 127.6, 120.5, 94.2, 75.2, 74.6, 67.7, 27.8, 27.3, 26, 23, 20.5, 18.6, -4.2, -5.0. HRMS: calcd for C24H42N5O4NaSi2 79Br [MNa+] 622.1856, obsd 622.1850. 5´,3´-O-Bis(t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-8-propargyloxyadenosine (4). Propargyloxy anion was generated in situ by allowing n-BuLi to react with excess anhydrous propargyl alcohol. 0.83 mmol (500 mg) of compound 4 was dissolved in 2 mL anhydrous THF in a round bottom flask under argon gas. Anhydrous propargyl alcohol (1 mL) was added to 4 mL freshly distilled THF in a reaction flask fitted with a balloon; the solution was kept under nitrogen atmosphere and cooled down to -40 °C. Then, 10 mmol n-BuLi (3.5 ml of 2.5M hexane solution) was gradually added to the reaction mixture; the by-product butane was collected in a balloon. The reaction was completed instantly. This in situ generated lithium propargyloxide was transferred to the flask containing 4 through an oven-dried syringe. The reaction was allowed to proceed for 20 h at room temperature. TLC monitoring indicated the absence of any 4 after that time period. The solution was neutralized by dilute acetic acid. Excess solvent was removed under reduced pressure and the solid residue was then partitioned between water and ethyl acetate. The ethyl acetate layer was collected and dried by using anhydrous Na2SO4. Flash chromatography was 59 used to elute (2:3 hexane:ethyl acetate) the desired compound, 4. Yield of 4 was 94%; silica gel TLC Rf 0.24 (2:3 hexane:ethyl acetate);1H NMR (300 MHz, CDCl3): δ 8.21 (s, 1H, 2-H), 5.93 (d, 1H, 1´-H), 5.61(s, 2H, NH2), 5.05-5.20 (m, 2H, CH2) 4.91-4.97 (q, 1H, 2´-H), 4.66-4.71 (d, 1H, 3´-H), 4.34-4.45 (m, 1H, 4´-H), 4.02-4.12 (m, 2H, 5´-H), 2.61-2.65 (t, 1H, CH), 1.00-1.20 (d, 18H, (CH3)3), 0.89 (s, 9H, (CH3)3), 0.11 (d, 6H, 2CH3). 13C NMR (75 MHz, CDCl3): δ 153.7, 153.2, 151.8, 149.8, 116.0, 90.5, 75.3, 74.8, 74.7, 67.9, 58.0, 27.8, 27.2, 26.1, 22.9, 20.7, 18.8, -4.2, -4.9. HRMS: calcd for C27H46N5O5Si2 [MH+] 576.3038, obsd 576.3030. 5´,3´-O-Bis(t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8-propargyl-oxyadenosine (5). The N6-amino group of 4 was protected using benzoyl chloride (BzCl). 0.77 mmol (435 mg) of 4 was dissolved in 2 mL anhydrous pyridine in a reaction flask and the solution was cooled down to -5 °C (by using salt-ice bath). Two equivalents of BzCl (1.54 mmol, 181μL) were added dropwise using a disposable syringe while stirring. The reaction mixture was allowed to warm to room temperature over a period of 1 h, and the reaction was continued for 4 h under argon. After that time, excess benzoyl chloride was quenched with 1 mL methanol. The reaction was allowed to stand for an h at 0 °C. Then, excess methanolic ammonia (1 mL 7N NH3) was added and the solution was stirred for an additional hour at the same temperature. The crude product mixture was then dried down under reduced pressure to a solid residue, which was first partitioned between ether and saturated NaHCO3 solution. The organic layer was then washed several times with water and, subsequently, dried with anhydrous Na2SO4. Product was then separated (3:1 hexane:ethyl acetate) by flash chromatography. Yield of the product 5 varied between 89% to 95%; silica gel TLC Rf 0.62 (2:3 hexane:ethyl 60 acetate); 1H NMR (300 MHz, CDCl3): δ 8.70-8.78 (bs, 1H, CONH-), 8.63 (s, 1H, 2-H), 7.95-8.02 (m, 2H, PhCO), 7.48-7.65 (m, 3H, PhCO), 5.91 (s, 1H, 1´-H), 5.13-5.29 (m, 2H, CH2), 4.86-4.95 (q, 1H, 2´-H), 4.68-4.74 (d, 1H, 3´-H), 4.35-4.47 (m, 1H, 4´-H), 4.03-4.16 (m, 2H, 5´-H), 2.60-2.63 (t, 1H, CH), 1.13 (s, 9H, (CH3)3), 1.05 (s, 9H, (CH3)3), 0.90 (s, 9H, (CH3)3), 0.07-0.15, (d, 6H, CH3). 13C NMR (75 MHz, CDCl3): δ 164.9, 154.9, 153.1, 151.3, 146.7, 134.2, 132.8, 129.1, 128.0, 120.5, 90.7, 75.5, 75.0, 67.8, 58.8, 27.8, 27.3, 26.1, 22.9, 20.6, 18.7, -4.2, -4.9. HRMS: calcd for C34H50N5O6Si2 [MH+] 680.3300, obsd 680.3297. 2´-O-t-Butyldimethylsilyl-N6-benzoyl-8-propargyloxyadenosine(6). Compound 5 was selectively deprotected at 5´-OH and 3´-OH by using (HF)x.py at subzero temperature. 0.74 mmol (505 mg) of 5 was added in CH2Cl2 and the solution was cooled down to -15 °C. 3.7 mmol (0.1 mL) of of (HF)x•py was diluted with 600 μL pyridine at 0 °C. The latter solution was then gradually added to the former solution and the reaction temperature was maintained at -15 °C. The reaction was complete in 2-2.5 h. 50 ml CH2Cl2 was added to the product mixture. The solution was then washed with saturated aqueous NaHCO3 solution. CH2Cl2 layer was then washed several times with water and dried with anhydrous Na2SO4. Pure product was obtained by flash chromatography (3:7 hexane:ethyl acetate). Yield of the desired product 6 was 82% to 95%; silica gel TLC Rf 0.13 (2:3 hexane:ethyl acetate);1H NMR (300 MHz, CDCl3): δ 8.99 (s, 1H, CONH-), 8.6(s, 1H, 2-H), 7.91-7.98 (m, 2H, PhCO), 7.42-7.58 (m, 3H, PhCO), 5.94-6.02 (d, 1H, 5´-OH), 5.89-5.93 (d, 1H, 1´-H), 5.12-5.16 (m, 2H, CH2), 5.03-5.09 (m, 1H, 2´-H), 4.27- 4.32 (m, 2H, 3´-H and 4´-H), 3.85-3.93 (bd, 1H, 5´-H), 3.64-3.74 (bt, 1H, 5´-H), 2.94 (s, 1H, 3´-OH), 2.60-2.64 (t, 1H, CH), 0.76 (s, 9H, (CH3)3), -0.18 (s, 3H, CH3), -0.37 (s, 3H, 61 CH3). 13C NMR (75 MHz, CDCl3): δ 164.7, 155.2, 150.9, 150.5, 147.4, 133.9, 132.7, 128.9, 127.8, 120.4, 87.9, 87.3, 76.3, 73.9, 72.7, 63.4, 58.4, 25.8, 17.8, -5.2, -5.3. HRMS: calcd for C26H34N5O6Si [MH+] 540.2278, obsd 540.2277. 5´-O-(4,4´-Dimethoxytrityl)-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8-proparg-yloxyadenosine (7). 0.204 mmol (110 mg) of compound 6 was dissolved in 2 mL anhydrous pyridine. The solution was cooled down to 0 °C under argon and 1.2 equivalent (85 mg) of DMT-Cl was added. The reaction mixture was stirred for 4 h at 0 °C and then for 6 h at room temperature. Then 1.2 equivalent of the DMT-Cl reagent was again added to the reaction mixture at 0 °C and the reaction was allowed to continue overnight. The reaction was quenched by addition of excess anhydrous methanol (0.5 mL). After another hour at room temperature, the solution was concentrated to dryness under reduced pressure. The crude solid was first fractioned between aqueous NaHCO3 and ethyl acetate, and then the organic layer was washed several times with water. A short chromatography column (2:3 hexane:ethyl acetate) was necessary to purify the product. Yield of the product 7 varied from 75% to 80%; silica gel TLC Rf 0.47 (2:3 hexane : ethyl acetate); 1H NMR (300 MHz, CDCl3): δ 8.91 (s, 1H, CONH-), 8.5 (s, 1H, 2-H), 7.94-8.02 (m, 2H, PhCO), 7.14-7.6 (m, 12H, Ph), 6.74-6.81 (dd, 4H, Ph), 5.97-6.03 (d, 1H, 1´-H), 5.0-5.20 (dq, 2H, CH2), 4.39-4.44 (t, 1H, 2´-H), 4.18-4.24 (q, 1H, 3´-H), 4.07-4.15 (q, 1H, 4´-H), 3.75 (s, 6H, OCH3), 3.45-3.52 (m, 1H, 5´-H), 3.28-3.37 (dd, 1H, 5´-H), 2.78-2.84 (d, 1H, 3´-OH), 2.54-2.58 (t, 1H, CH), 0.83 (s, 9H, (CH3)3), -0.01 (s, 3H, CH3), -0.17 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3): δ 165, 158.6, 155.7, 152.4, 151.2, 146.8, 145.1, 136.3, 136.1, 134.3, 132.8, 130-130.4 (d), 129.1, 128.4, 128, 127, 62 120, 113, 87.2, 86.5, 84, 72.8, 71.6, 63.8, 58.6, 55.4, 25.8, 18, 14.4, -4.6, -4.8. HRMS: calcd for C47H51N5O8SiNa [MNa+] 864.3405, obsd 864.3405. 5´-O-(4,4´-Dimethoxytrityl)-3´-O-[(2-cyanoethoxy)(N,N-diisopropylamino)ph-osphino]- 2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8-propargyloxyadenosine (8). 8- Propargyloxyadenosine phosphoramidite was synthesized using the 2-cyanoethyl N,N-diisopropylchlorophosphoramidite reagent. 0.178 mmol (150 mg) of compound 7 (was dissolved in 5 mL of freshly distilled THF contained in an oven-dried flask. Five equivalent (0.9 mmol, 205 μL) DIPEA was added and the solution was stirred for 5 min at room temperature (23 °C), and then cooled down to 0 °C, under argon atmosphere. 81 μL (0.36 mmol, 2 equivalent) of phosphoramidite was added dropwise into the flask. Reaction was stirred at 0 °C for 30 min and then gradually warmed up to room temperature. After another hour under inert atmosphere, the solvent was evaporated by rotary evaporation and the desired product was separated by silica gel column chromato-graphy (11:9 hexane:ethyl acetate). Yield of 8 varied from 82% to 90%; silica gel TLC Rf 0.47 (2:3 hexane:ethyl acetate); 1H NMR (300 MHz, CDCl3): 8.76-8.82 (d, 1H, CONH-), 8.44-8.52 (d, 1H, 2-H), 7.94-8.02 (m, 2H, PhCO), 7.14-7.62 (m, 12H, Ph), 6.74-6.82 (m, 4H, Ph), 5.95-6.02 (t, 1H, 1´-H), 5.29-5.40 (m, 1H, 2´-H), 5.02-5.4 (m, 2H, CH2), 4.3- 4.55 (m, 2H, 3´-H and 4´-H), 4.07-4.24 (q, 1H, 5´-H), 3.78-4.02 (m, 1H, 5´-H), 3.75 (s, 6H, OCH3), 3.51-3.68 (m, 4H, CH2CN, 2CHN), 2.64-2.70 (m,1H,P-O-CH2), 2.50-2.56 (t, 1H, CH), 2.28-2.34 (m, 1H, P-O-CH2), 1.14-1.3 (m, 12H, 4CH3), 0.70-0.80 (d, 9H, (CH3)3), (-0.08)-0.0 (d, 3H, CH3),(-0.34)-(-0.20) (s, 3H, CH3). 13C NMR (75 MHz, CDCl3): δ 164.8, 158.6, 156, 155.9, 152.4, 152.3, 151.2, 146.8, 145.1, 144.9, 136.4- 136.1(m), 134.4, 132.8, 130.4, 129.1, 128.5, 127, 120.1, 117.6, 117.9, 113.3, 86.4-87.0 63 (q), 84.0 (d), 72-73.8 (m), 63.4-63.8(d), 60.8, 59.0-59.4, 58.4, 57.8-58.4, 55.5, 43.5-43.8 (d), 43.0-43.4 (m), 25.9, 24.7-25.2 (m), 22.8, 23.4, 21.4, 20.7, 20.3, 18.2, 14.5, -4.3, -4.8. 31P NMR (CD2Cl2): 149.7, 151.8. HRMS: calcd for C56H68N7O9SiPNa [MNa+] 1064.4483, obsd 1064.4493. All phenylethoxyadenosine and cyclohexylethoxyadenosine derivatives were prepared following procedures analogous to those used for the propargyloxyadenosine derivatives. 5´,3´-O-Bis(t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-8-phenylethoxyadenosine (9). Yield 97%; silica gel TLC Rf 0.32 (1:1 hexane:ethyl acetate); 1H NMR (300 MHz, CDCl3): δ 8.19 (s, 1H, 2-H), 7.23-7.36 (m, 5H, Ph), 5.85 (s, 1H, 1´-H), 5.52 (s, 2H, NH2), 4.90-4.98 (q, 1H, 2´-H), 4.67-4.76 (m, 3H, 3´-H, OCH2), 4.34-4.41 (m, 1H, 4´-H), 3.95- 4.10 (m, 2H, 5´-H), 3.12-3.2 (t, 2H, CH2), 1.00-1.16 (d, 18H, (CH3)3), 0.87 (s, 9H, (CH3)3), 0.02-.10 (d, 6H, 2CH3). 13C NMR (75 MHz, CDCl3): δ 154.2, 153.4, 151.5, 149.8, 116.0, 90.5, 75.5, 74.6, 71.1, 67.9, 35.2, 27.6, 27.2, 26.1, 22.9, 20.7, 18.6, -4.2, - 4.9. 5´,3´-O-Bis(t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8-phenylethox-yadenosine (10). Yield 80-92%; silica gel TLC Rf 0.67 (2:3 hexane:ethyl acetate); 1H NMR (300 MHz, CDCl3): δ 8.80-8.88 (bs, 1H, CONH-), 8.59 (s, 1H, 2-H), 7.94-8.02 (m, 2H, PhCO), 7.46-7.62 (m, 3H, PhCO), 7.24-7.36 (m, 5H, Ph), 5.91 (s, 1H, 1´-H), 4.88- 4.96 (dd, 1H, 2´-H), 4.76-4.84 (t, 2H, CH2), 4.68-4.74 (d, 1H, 3´-H), 4.35-4.42 (q, 1H, 4´- H), 3.95-4.13 (m, 2H, 5´-H), 3.13-3.22 (t, 2H, CH2), 1.2 (s, 9H, (CH3)3), 1.05 (s, 9H, (CH3)3), 0.89 (s, 9H, (CH3)3), 0.03-0.11 (d, 6H, 2CH3). 13C NMR (75 MHz, CDCl3): δ 165, 156, 152, 151, 146.4, 137, 134.3, 132.8, 129, 128.0, 127 121, 90.6, 75.5, 74.5, 71.8, 64 67.8, 35.2, 27.8, 27.3, 26.1, 23, 20.7, 18.7, -4.2, -4.9. HRMS: calcd for C39H55N5O6NaSi2 [MNa+] 768.3589, obsd 768.3600. 2´-O-t-Butyldimethylsilyl-N6-benzoyl-8-phenylethoxyadenosine (11). Yield 88- 94%; silica gel TLC Rf 0.12 (2:3 hexane:ethyl acetate); 1H NMR (300 MHz, CDCl3): δ 8.88 (bs, 1H, CONH-), 8.62 (s, 1H, 2-H), 7.94-8.0 (m, 2H, PhCO), 7.44-7.62 (m, 3H, PhCO), 7.22-7.34 (m, 5H, Ph), 5.98-6.06 (d, 1H, 5´-OH), 5.87-5.92 (d, 1H, 1´-H), 5.07- 5.14 (m, 1H, 2´-H), 4.62-4.82 (m, 2H, CH2), 4.29-4.35 (d, 2H, 5´-H), 4.05-4.13 (q, 1H, 3´-H), 3.87-3.97 (d, 1H, 4´-H), 3.10-3.18 (t, 2H, CH2), 2.90 (s, 1H, 3´-OH), 0.77 (s, 9H, (CH3)3), -0.16 (s, 3H, CH3), -0.40 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3): δ 164.7, 156.2, 151, 150.5, 147.2, 136.9, 134.2, 133, 129, 127.2, 121, 87.4-88 (d), 72-74 (t), 35.4, 25.7, 17.8, -5.0, -5.3. HRMS: calcd for C31H39N5O6NaSi [MNa+] 628.2567, obsd 628.2575. 5´-O-(4,4´-Dimethoxytrityl)-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8-phenyl-ethoxyadenosine (12). Yield 68-81%; silica gel TLC Rf 0.6 (2:3 hexane:ethyl acetate); 1H NMR (300 MHz, CDCl3): δ 8.68 (bs, 1H, CONH-), 8.47 (s, 1H, 2-H), 7.94-8.02 (m, 2H, PhCO), 7.42-7.62 (m, 5H, Ph), 7.14-7.38 (m, 12H, Ph), 6.72-6.8 (dd, 4H, Ph), 5.87- 5.92 (d, 1H, 1´-H), 5.26-5.32 (m, 1H, 2´-H), 4.66-4.80 (M, 2H, CH2), 4.32-4.4.38 (q, 1H, 3´-H), 4.16-4.22 (q, 1H, 4´-H), 3.75 (s, 6H, OCH3), 3.28-3.5 (m, 2H, 5´-H), 3.04-3.12 (t, 2H, CH2), 2.71-2.75 (d, 1H, 3´-OH), 0.80 (s, 9H, (CH3)3), -0.07 (s, 3H, CH3), -0.23 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3): δ 171.3, 164.9, 158.5, 156.5, 152.2, 151, 146.3, 145.1, 136.9, 136.1, 136.2, 134.4, 132.8, 130.3, 128.9-129.1 (d), 128.5, 127.1, 127, 121, 113, 87.2, 86.4, 84, 72.5, 71.8, 71.6, 63.9, 60.7, 55.4, 35.3, 25.8, 21.4 18, 14.5, -4.78, - 4.88 HRMS: calcd for C52H57N5O8NaSi [MNa+] 930.3874, obsd 930.3867. 65 5´-O-(4,4´-Dimethoxytrityl)-3´-O-[(2-cyanoethoxy)(N,N-diisopropylamino)pho-sphino]- 2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8-phenylethoxyadenosine(13). Yield 82-86% silica gel TLC Rf 0.6 (2:3 hexane:ethyl acetate); 1H NMR (300 MHz, CDCl3): 8.66 (s, 1H, CONH-), 8.40-8.46 (d, 1H, 2-H), 7.96-8.02 (m, 2H, PhCO), 7.42-7.62 (m, 5H, Ph), 7.20-7.39 (m, 12H, Ph), 6.74-6.82 (m, 4H, Ph), 5.88-5.94 (t, 1H, 1´-H), 5.36- 5.46 (m, 1H, 2´-H), 4.68-4.78 (m, 2H, OCH2), 4.30-4.54 (m, 2H, 3´-H and 4´-H), 3.82- 4.02 (m, 1H, 5´-H), 3.76 (s, 6H, OCH3), 3.51-3.70 (m, 4H, CH2CN, 2CHN), 3.24-3.34 (m, 1H, 5´-H), 3.09-3.16 (t, 2H, CH2), 2.51-2.70 (m, 2H, P-O-CH2), 1.08-1.32 (m, 12H, 4CH3), 0.72-0.77 (d, 9H, (CH3)3), (-0.09)-(-0.04) (d, 3H, CH3), (-0.33)-(-0.28) (d, 3H, CH3). 13C NMR (75 MHz, CDCl3): δ 164.5, 158.6, 156.8, 155.9, 152.4, 146.4, 145.0, 144.9, 137, 136, 134.5, 132.8, 130.4, 129.3, 129, 128.6, 128, 127, 117.6, 113.3, 98.8, 87.0, 86.5, 83.9, 73, 71.2, 63.5, 59.0-59.4 (d), 55.5, 43.5-43.8 (d), 35.5, 25.8, 24.7-25.2 (m), 21.4, 20.7, 18.2, 14.5, -4.4, -5.0. 31P NMR (CD2Cl2): 149.0, 151.4. HRMS: calcd for C61H74N7O9NaSiP [MNa+] 1130.4953, obsd 1130.4973. 5´,3´-O-Bis(t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-8-cyclohexylethoxyaden-osine (14). Yield 65%; silica gel TLC Rf 0.49 (2:3 hexane:ethyl acetate); 1H NMR (300 MHz, CDCl3): δ 8.20 (s, 1H, 2-H), 5.89 (d, 1H, 1´-H), 5.34(s, 2H, NH2), 4.92-4.97 (m, 1H, 2´-H), 4.68-4.74 (d, 1H, 3´-H), 4.47-4.61 (m, 2H, OCH2), 4.34-4.44 (m, 1H, 4´-H), 3.98-4.12 (m, 2H, 5´-H), 1.16-1.82 (m, 13H, C6H11CH2), 1.02-1.16 (d, 18H, (CH3)3), 0.89 (s, 9H, (CH3)3), 0.04-.12 (d, 6H, 2CH3). 13C NMR (75 MHz, CDCl3): δ 154.4, 153.2, 151.4, 149.6, 116.3, 90.4, 75.4, 74.6-74.8, 67.2, 67.8, 36.4, 33.3, 27.8, 27.3, 26.6, 26.3, 26.1, 22.9, 20.5, 18.6, -4.3, -4.9. HRMS: calcd for C32H58N5O5Si2 [MH+] 648.3977, obsd 648.4002. 66 5´,3´-O-Bis(t-butylsilyl)-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8-cyclohexylet-hoxyadenosine (15). Yield 84-92%; silica gel TLC Rf 0.73 (2:3 hexane:ethyl acetate); 1H NMR (300 MHz, CDCl3): δ 8.76-8.82 (bs, 1H, CONH-), 8.59 (s, 1H, 2-H), 7.95-8.02 (m, 2H, PhCO), 7.48-7.62 (m, 3H, PhCO) 5.91-5.95 (d, 1H, 1´-H), 4.88-4.97 (q, 1H, 2´-H), 4.70-4.75 (d, 1H, 3´-H), 4.53-4.69 (m, 2H, OCH2), 4.35-4.43 (q, 1H, 4´-H), 4.98-4.14 (m, 2H, 5´-H), 1.16-1.82 (m, 13H, C6H11CH2), 1.13 (s, 9H, (CH3)3), 1.05 (s, 9H, (CH3)3), 0.89 (s, 9H, (CH3)3), 0.06-0.14, (d, 6H, CH3). 13C NMR (75 MHz, CDCl3): δ 165, 156.1, 152.1, 150.5, 146, 134.2, 132.7, 129.0, 128.0, 121.0, 90.5, 75.4, 74.8, 70.0, 67.7, 36.3, 33.3, 27.8, 27.3, 26.6, 26.3, 26.1, 22.9, 20.6, 18.7, -4.1, -5.0. HRMS: C39H61N5O6NaSi2 [MNa+] 774.4058, obsd 774.4078. 2´-O-t-Butyldimethylsilyl-N6-benzoyl-8-cyclohexylethoxyadenosine(16). Yield 87-90%; silica gel TLC Rf 0.16 (2:3 hexane:ethyl acetate); 1H NMR (300 MHz, CDCl3): δ 8.79 (s, 1H, CONH-), 8.64 (s, 1H, 2-H), 7.94-8.00 (m, 2H, PhCO), 7.47-7.60 (m, 3H, PhCO), 6.04-6.12 (d, 1H, 5´-OH), 5.88-5.92 (d, 1H, 1´-H), 5.08-5.16 (q, 1H, 2´-H), 4.48- 4.64 (m, 2H, OCH2), 4.30-4.36 (m, 2H, 3´-H and 4´-H), 3.88-3.98 (d, 1H, 5´-H), 3.67- 3.78 (t, 1H, 5´-H), 2.90 (s, 1H, 3´-OH), 0.9-1.80 (m, 13H, C6H11CH2), 0.80 (s, 9H, (CH3)3), -0.14 (s, 3H, CH3), -0.37 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3): δ 164.7, 156.6, 151.0, 150.4, 147.0, 134.3, 132.9, 129.0, 127.0, 120.9, 88.0, 87.4, 73.9, 73.0, 70.2, 63.7, 36.3, 34.5, 33.4, 26.6, 26.3, 25.7, 18.0, -5.0, -5.2. HRMS: calcd for C31H46N5O6Si [MH+] 612.3217, obsd 612.3228. 5´-O-(4,4´-Dimethoxytrityl)-2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8-cyclohex-ylethoxyadenosine (17). Yield 74-78%; silica gel TLC Rf 0.59 (2:3 hexane:ethyl acetate); 1H NMR (300 MHz, CDCl3): δ 8.70 (s, 1H, CONH-), 8.46 (s, 1H, 2-H), 7.96- 67 8.02 (m, 2H, PhCO), 7.14-7.62 (m, 12H, Ph), 6.72-6.80 (dd, 4H, Ph), 5.88-5.93 (d, 1H, 1´-H), 5.33-5.39 (t, 1H, 2´-H), 4.53-4.62 (t, 2H, OCH2), 4.38-4.44 (q, 1H, 3´-H), 4.16- 4.23 (q, 1H, 4´-H), 3.76 (s, 6H, OCH3), 3.30-3.50 (m, 2H, 5´-H), 2.73-2.77 (d, 1H, 3´- OH), 0.92-1.80 (m, 13H, C6H11CH2), 0.84 (s, 9H, (CH3)3), -0.02 (s, 3H, CH3), -0.2 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3): δ 164.8, 158.6, 156.8, 152.0, 150.8, 146.3, 145.0, 136.3, 136.2, 134.5, 132.7, 130-130.4 (d), 129.1, 128.5, 127.9, 126.9, 120.5, 113.2, 87.3, 86.4, 84.1, 72.7, 71.7, 70.0, 63.9, 55.4, 36.3, 34.5, 33.4, 33.3, 26.8, 26.4, 25.8, 18.1, -4.7, -4.8 HRMS: calcd for C52H63N5O8NaSi [MNa+] 936.4344, obsd 936.4332. 5´-O-(4,4´-Dimethoxytrityl)-3´-O-[(2-cyanoethoxy)(N,N-diisopropylamino)ph-osphino]- 2´-O-(t-butyldimethylsilyl)-N6-benzoyl-8-cyclohexylethoxyadenosine (18). Yield 78-85%; silica gel TLC Rf 0.59 (2:3 hexane:ethyl acetate); 1H NMR (300 MHz, CDCl3): 8.64-8.72 (d, 1H, CONH-), 8.35-8.44 (t, 1H, 2-H), 7.96-8.02 (m, 2H, PhCO), 7.16-7.62 (m, 12H, Ph), 6.74-6.82 (m, 4H, Ph), 5.86-5.94 (t, 1H, 1´-H), 5.35-5.48 (m, 1H, 2´-H), 4.53-4.62 (t, 2H, OCH2), 4.44-4.53 (m, 1H, 3´-H), 4.24-4.40 (m, 1H, 4´-H), 3.88- 4.0 (m, 1H, 5´-H), 3.77 (s, 6H, OCH3), 3.51-3.70 (m, 4H, CH2CN, 2CHN), 3.24-3.38 (m, 1H, 5´-H), 2.54-2.74 (m, 2H, P-O-CH2), 0.84-1.80 (m, 13H, C6H11CH2; m, 12H, 4CH3), 0.72-0.80 (d, 9H, (CH3)3), (-0.08)-(-0.04) (t, 3H, CH3), (-0.30)-(-0.26) (d, 3H, CH3). 13C NMR (75 MHz, CDCl3): δ 164.8, 158.6, 150.7, 145.0, 136.2, 132.8, 130.4, 129.1, 128.5, 128.0, 127.0, 120.1, 113.2, 86.4-87.0 (q), 83.0, 70.0, 63.8, 59.0, 59.4, 55.3, 53.5, 43-43.4 (d), 36.3, 34.5, 33.2, 33.4, 26.6, 26.4, 25.8, 24.6-25.1 (m), 20.5-20.8, 18.1, -4.3, -4.9. 31P NMR (CD2Cl2): 148.9, 151.5. HRMS: calcd for C61H80N7O9NaSiP [MNa+] 1136.5422, obsd 1136.5436. 68 Design of the caspase 2 siRNA and synthesis of the modified guide strands. Caspase 2 siRNA (A:U) and the corresponding negative control siRNA (scrambled sequence) was designed using Ambion's siRNA designing tool and checked for sequence similarity using nucleotide Basic Local Alignment Search Tool (BLAST). No significant similarity was found with other genes. To analyze caspase 2 expression levels quickly and reliably, a plasmid-based dual luciferase assay system (psiCHECK2 vector) was employed. A fragment of the caspase 2 mRNA sequence was inserted into the vector, and the resulting reporter plasmid was used to evaluate caspase 2 mRNA knockdown. 8-AlkoxyA phosphoramidites was subsequently coupled into various positions of the guide strand of the caspase 2 siRNA. All siRNA oligonucleotides, modified and unmodified, were synthesized by standard solid-phase RNA synthesis on DNA columns in the DNA/Peptide Core Facility of the University of Utah using an Applied Biosystems (Model 394) DNA/RNA synthesizer. The positions chosen for single substitution were 4, 6, 10 and 15; doubly modified guide strands had modifications at (6,10), (6,15) and (10, 15) positions (Figure 2.10). Synthesis and purification of siRNAs. After synthesis, the 21-mer RNA oligonucleotides were cleaved from the column and deprotected using methanolic ammonia for 24 h at room temperature, and the 2´-OTBDMS group was deprotected by using TEA•3HF overnight at room temperature. The oligomers were then dialyzed at 4 °C for 6 h, purified by semi-preparative ion-exchange HPLC, dialyzed at 4 °C again to get rid of excess salt, lyophilized and stored at -20 °C under dry conditions. 69 Figure 2.10. SiRNA sequences used. A:U represents unmodified siRNA and modified (denoted by M:G) sequences have the standard A:U base pair replaced by 8- alkoxyA(M):G in one or two positions in the siRNAs. A:U M4:G M6:G M10:G M15:G M(6,10):G M(6,15):G M(10,15):G passenger: 5'-CCCUUCUGCUUUCUAUUACdTdT-3' guide: 3'-dTdTGGGAAGACGAAAGAUAAUG-5' passenger: 5'-CCCUUCUGCUUUCUAGUACdTdT-3' guide: 3'-dTdTGGGAAGACGAAAGAUMAUG-5' passenger: 5'-CCCUUCUGCUUUCGAUUACdTdT-3' guide: 3'-dTdTGGGAAGACGAAAGMUAAUG-5' passenger: 5'-CCCUUCUGCGUUCUAUUACdTdT-3' guide: 3'-dTdTGGGAAGACGMAAGAUAAUG-5' passenger: 5'-CCCUGCUGCUUUCUAUUACdTdT-3' guide: 3'-dTdTGGGAMGACGAAAGAUAAUG-5' passenger: 5'-CCCUUCUGCGUUCGAUUACdTdT-3' guide: 3'-dTdTGGGAAGACGMAAGMUAAUG-5' passenger: 5'-CCCUGCUGCUUUCGAUUACdTdT-3' guide: 3'-dTdTGGGAMGACGAAAGMUAAUG-5' passenger: 5'-CCCUGCUGCGUUCUAUUACdTdT-3' guide: 3'-dTdTGGGAMGACGMAAGAUAAUG-5' 15 10 6 4 N N N N NH2 O R M = 8-PgOA (8-propargyloxyA) 8-PeOA (8-phenylethoxyA) 8-CeOA (8-cyclohexylethoxyA ) M4 = Guide strand containing M at position 4 from the 5'-end of the strand R 70 ESI-MS characterization of the modified guide strands. Two nanomoles of each modified guide strands were extensively dialyzed for 2 days at 4 °C in ammonium acetate solution to get rid of excess sodium ions. The final dialysis was carried out in water. The oligomers were then lyophilized, and dissolved in 1:1 isopropanol:water and analyzed by electrospray ionization mass spectrometry (ESI-MS) in negative ion mode. The results are furnished in the Table 2.1. Results and discussion Purines can exist preferentially as the anti conformer and participate in canonical Watson-Crick H-bonding with their complementary bases. However, there is evidence of transient syn adenosines in duplex RNA (24). Also, in the context of AG mismatches, there is evidence of syn A in double-stranded DNA (25). It was observed that the syn/anti equilibrium of purines could be shifted more towards the syn conformation by introducing a substitution at position 8; this substituent experiences a steric clash with the 5´ hydrogen atoms of the ribose backbone as well as with the 4´ oxygen, facilitating the syn conformation to a greater extent than the unmodified purine. Indeed, 8-BrdG, 8-O-dA, 8-O-dG, 8-BrdA and 8-MeOdA are known to exist in mixtures of syn/anti equilibrium (26-29). Here, the unusual RNA base modification 8- alkoxyA was chosen as a potential ‘conformational switch' in the guide strand of a caspase 2 siRNA. 8-AlkoxyA, being a Janus-faced base, can expose the Watson-Crick face in the anti conformation, as well as the Hoogsteen face in the syn conformation; thus anti 8-alkoxyA can base-p