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Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme.

Li F, Pallan PS, Maier MA, Rajeev KG, Mathieu SL, Kreutz C, Fan Y, Sanghvi J, Micura R, Rozners E, Manoharan M, Egli M
Nucleic Acids Res 35 6424-38 (2007)
Cited: 35 times
EuropePMC logo PMID: 17881374

Abstract

Short interfering RNA (siRNA) duplexes are currently being evaluated as antisense agents for gene silencing. Chemical modification of siRNAs is widely expected to be required for therapeutic applications in order to improve delivery, biostability and pharmacokinetic properties. Beyond potential improvements in the efficacy of oligoribonucleotides, chemical modification may also provide insight into the mechanism of mRNA downregulation mediated by the RNA-protein effector complexes (RNA-induced silencing complex or RISC). We have studied the in vitro activity in HeLa cells of siRNA duplexes against firefly luciferase with substitutions in the guide strand of U for the apolar ribo-2,4-difluorotoluyl nucleotide (rF) [Xia, J. et al. (2006) ACS Chem. Biol., 1, 176-183] as well as of C for rF. Whereas an internal rF:A pair adjacent to the Ago2 ('slicer' enzyme) cleavage site did not affect silencing relative to the native siRNA duplex, the rF:G pair and other mismatches such as A:G or A:A were not tolerated. The crystal structure at atomic resolution determined for an RNA dodecamer duplex with rF opposite G manifests only minor deviations between the geometries of rF:G and the native U:G wobble pair. This is in contrast to the previously found, significant deviations between the geometries of rF:A and U:A pairs. Comparison between the structures of the RNA duplex containing rF:G and a new structure of an RNA with A:G mismatches with the structures of standard Watson-Crick pairs in canonical duplex RNA leads to the conclusion that local widening of the duplex formed by the siRNA guide strand and the targeted region of mRNA is the most likely reason for the intolerance of human Ago2 (hAgo2), the RISC endonuclease, toward internal mismatch pairs involving native or chemically modified RNA. Contrary to the influence of shape, the thermodynamic stabilities of siRNA duplexes with single rF:A, A:A, G:A or C:A (instead of U:A) or rF:G pairs (instead of C:G) show no obvious correlation with their activities. However, incorporation of three rF:A pairs into an siRNA duplex leads to loss of activity. Our structural and stability data also shed light on the role of organic fluorine as a hydrogen bond acceptor. Accordingly, UV melting (T(M)) data, osmotic stress measurements, X-ray crystallography at atomic resolution and the results of semi-empirical calculations are all consistent with the existence of weak hydrogen bonds between fluorine and the H-N1(G) amino group in rF:G pairs of the investigated RNA dodecamers.

Articles - 2q1r mentioned but not cited (4)

  1. Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme. Li F, Pallan PS, Maier MA, Rajeev KG, Mathieu SL, Kreutz C, Fan Y, Sanghvi J, Micura R, Rozners E, Manoharan M, Egli M. Nucleic Acids Res 35 6424-6438 (2007)
  2. A new way to see RNA. Keating KS, Humphris EL, Pyle AM. Q Rev Biophys 44 433-466 (2011)
  3. Sequence dependent variations in RNA duplex are related to non-canonical hydrogen bond interactions in dinucleotide steps. Kailasam S, Bhattacharyya D, Bansal M. BMC Res Notes 7 83 (2014)
  4. Towards a comprehensive understanding of RNA deamination: synthesis and properties of xanthosine-modified RNA. Mair S, Erharter K, Renard E, Brillet K, Brunner M, Lusser A, Kreutz C, Ennifar E, Micura R. Nucleic Acids Res 50 6038-6051 (2022)


Reviews citing this publication (11)

  1. Carbon-oxygen hydrogen bonding in biological structure and function. Horowitz S, Trievel RC. J Biol Chem 287 41576-41582 (2012)
  2. Exploring chemical modifications for siRNA therapeutics: a structural and functional outlook. Shukla S, Sumaria CS, Pradeepkumar PI. ChemMedChem 5 328-349 (2010)
  3. Chemical RNA modifications for studies of RNA structure and dynamics. Wachowius F, Höbartner C. Chembiochem 11 469-480 (2010)
  4. Chemical modification of siRNA bases to probe and enhance RNA interference. Peacock H, Kannan A, Beal PA, Burrows CJ. J Org Chem 76 7295-7300 (2011)
  5. Novel modifications in RNA. Phelps K, Morris A, Beal PA. ACS Chem Biol 7 100-109 (2012)
  6. Delivery of RNAi therapeutics: work in progress. Miller AD. Expert Rev Med Devices 10 781-811 (2013)
  7. Clinical potential of oligonucleotide-based therapeutics in the respiratory system. Moschos SA, Usher L, Lindsay MA. Pharmacol Ther 169 83-103 (2017)
  8. Crystallographic studies of chemically modified nucleic acids: a backward glance. Egli M, Pallan PS. Chem Biodivers 7 60-89 (2010)
  9. Beyond ribose and phosphate: Selected nucleic acid modifications for structure-function investigations and therapeutic applications. Liczner C, Duke K, Juneau G, Egli M, Wilds CJ. Beilstein J Org Chem 17 908-931 (2021)
  10. The steric hypothesis for DNA replication and fluorine hydrogen bonding revisited in light of structural data. Egli M. Acc Chem Res 45 1237-1246 (2012)
  11. Computational approaches to predicting the impact of novel bases on RNA structure and stability. Harrison JG, Zheng YB, Beal PA, Tantillo DJ. ACS Chem Biol 8 2354-2359 (2013)

Articles citing this publication (20)

  1. Unexpected origins of the enhanced pairing affinity of 2'-fluoro-modified RNA. Pallan PS, Greene EM, Jicman PA, Pandey RK, Manoharan M, Rozners E, Egli M. Nucleic Acids Res 39 3482-3495 (2011)
  2. 2'-Azido RNA, a versatile tool for chemical biology: synthesis, X-ray structure, siRNA applications, click labeling. Fauster K, Hartl M, Santner T, Aigner M, Kreutz C, Bister K, Ennifar E, Micura R. ACS Chem Biol 7 581-589 (2012)
  3. Chemical synthesis of site-specifically 2'-azido-modified RNA and potential applications for bioconjugation and RNA interference. Aigner M, Hartl M, Fauster K, Steger J, Bister K, Micura R. Chembiochem 12 47-51 (2011)
  4. Modulation of thermal stability can enhance the potency of siRNA. Addepalli H, Meena, Peng CG, Wang G, Fan Y, Charisse K, Jayaprakash KN, Rajeev KG, Pandey RK, Lavine G, Zhang L, Jahn-Hofmann K, Hadwiger P, Manoharan M, Maier MA. Nucleic Acids Res 38 7320-7331 (2010)
  5. Hydration changes upon DNA folding studied by osmotic stress experiments. Nakano S, Yamaguchi D, Tateishi-Karimata H, Miyoshi D, Sugimoto N. Biophys J 102 2808-2817 (2012)
  6. Effects of N2,N2-dimethylguanosine on RNA structure and stability: crystal structure of an RNA duplex with tandem m2 2G:A pairs. Pallan PS, Kreutz C, Bosio S, Micura R, Egli M. RNA 14 2125-2135 (2008)
  7. Structure and activity of Y-class DNA polymerase DPO4 from Sulfolobus solfataricus with templates containing the hydrophobic thymine analog 2,4-difluorotoluene. Irimia A, Eoff RL, Pallan PS, Guengerich FP, Egli M. J Biol Chem 282 36421-36433 (2007)
  8. Pairing geometry of the hydrophobic thymine analogue 2,4-difluorotoluene in duplex DNA as analyzed by X-ray crystallography. Pallan PS, Egli M. J Am Chem Soc 131 12548-12549 (2009)
  9. Short interfering RNA guide strand modifiers from computational screening. Onizuka K, Harrison JG, Ball-Jones AA, Ibarra-Soza JM, Zheng Y, Ly D, Lam W, Mac S, Tantillo DJ, Beal PA. J Am Chem Soc 135 17069-17077 (2013)
  10. High-resolution NMR analysis of the conformations of native and base analog substituted retroviral and LTR-retrotransposon PPT primers. Yi-Brunozzi HY, Brinson RG, Brabazon DM, Lener D, Le Grice SF, Marino JP. Chem Biol 15 254-262 (2008)
  11. Steric effects in RNA interference: probing the influence of nucleobase size and shape. Somoza A, Silverman AP, Miller RM, Chelliserrykattil J, Kool ET. Chemistry 14 7978-7987 (2008)
  12. Thioguanosine Conversion Enables mRNA-Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC-seq DUAL). Gasser C, Delazer I, Neuner E, Pascher K, Brillet K, Klotz S, Trixl L, Himmelstoß M, Ennifar E, Rieder D, Lusser A, Micura R. Angew Chem Int Ed Engl 59 6881-6886 (2020)
  13. Molecular dynamics simulation and binding energy calculation for estimation of oligonucleotide duplex thermostability in RNA-based therapeutics. Shen L, Johnson TL, Clugston S, Huang H, Butenhof KJ, Stanton RV. J Chem Inf Model 51 1957-1965 (2011)
  14. Overcoming GNA/RNA base-pairing limitations using isonucleotides improves the pharmacodynamic activity of ESC+ GalNAc-siRNAs. Schlegel MK, Matsuda S, Brown CR, Harp JM, Barry JD, Berman D, Castoreno A, Schofield S, Szeto J, Manoharan M, Charissé K, Egli M, Maier MA. Nucleic Acids Res 49 10851-10867 (2021)
  15. Promiscuous 8-alkoxyadenosines in the guide strand of an siRNA: modulation of silencing efficacy and off-pathway protein binding. Ghanty U, Fostvedt E, Valenzuela R, Beal PA, Burrows CJ. J Am Chem Soc 134 17643-17652 (2012)
  16. Short RNA duplexes guide sequence-dependent cleavage by human Dicer. Bergeron L, Perreault JP, Abou Elela S. RNA 16 2464-2473 (2010)
  17. Determination of nucleic acid hydration using osmotic stress. Rozners E. Curr Protoc Nucleic Acid Chem Chapter 7 Unit 7.14 (2010)
  18. Fluorobenzene Nucleobase Analogues for Triplex-Forming Peptide Nucleic Acids. Kumar V, Rozners E. Chembiochem 23 e202100560 (2022)
  19. Synthesis, physicochemical and biological properties of oligonucleotides incorporated with amino-isonucleosides. Wang F, Chen Y, Huang Y, Jin HW, Zhang LR, Yang ZJ, Zhang LH. Sci China Chem 55 70-79 (2012)
  20. Anionic G•U pairs in bacterial ribosomal rRNAs. Westhof E, Watson ZL, Zirbel CL, Cate JHD. RNA 29 1069-1076 (2023)


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