ACS Publications. Most Trusted. Most Cited. Most Read
My Activity
Recently Viewed
You have not visited any articles yet, Please visit some articles to see contents here.
CONTENT TYPES

Figure 1Loading Img

Structural Insights into DNA Replication without Hydrogen Bonds

View Author Information
Departments of Chemistry and Biology, Konstanz Research School Chemical Biology, Universität Konstanz, Universitätsstrasse 10, D-78464 Konstanz, Germany
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
Cite this: J. Am. Chem. Soc. 2013, 135, 49, 18637–18643
Publication Date (Web):November 27, 2013
https://doi.org/10.1021/ja409609j
Copyright © 2013 American Chemical Society
Article Views
3197
Altmetric
-
Citations
LEARN ABOUT THESE METRICS
Read OnlinePDF (3 MB)
Supporting Info (1)»

Abstract

Abstract Image

The genetic alphabet is composed of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chemical potential of DNA. d5SICS-dNaM is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq DNA polymerase revealed that the insertion of d5SICSTP opposite dNaM proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson–Crick-like structure. To understand the remaining steps of replication, we now report the characterization of the prechemistry complexes corresponding to the insertion of dNaMTP opposite d5SICS, as well as multiple postchemistry complexes in which the already formed unnatural base pair is positioned at the postinsertion site. Unlike with the insertion of d5SICSTP opposite dNaM, addition of dNaMTP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the postinsertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are observed, depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by deintercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation.

Supporting Information

ARTICLE SECTIONS
Jump To

Supporting table and figures, crystallization conditions, and oligonucleotide sequences. This material is available free of charge via the Internet at http://pubs.acs.org.

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Cited By


This article is cited by 63 publications.

  1. Jialu Zhang, Yihao Huang, Miao Sun, Shuang Wan, Chaoyong Yang, Yanling Song. Recent Advances in Aptamer-Based Liquid Biopsy. ACS Applied Bio Materials 2022, 5 (5) , 1954-1979. https://doi.org/10.1021/acsabm.1c01202
  2. Honglei Wang, Luying Wang, Nana Ma, Wuyuan Zhu, Bianbian Huo, Anlian Zhu, Lingjun Li. Access to Photostability-Enhanced Unnatural Base Pairs via Local Structural Modifications. ACS Synthetic Biology 2022, 11 (1) , 334-342. https://doi.org/10.1021/acssynbio.1c00451
  3. Lukas Eberlein, Frank R. Beierlein, Nico J. R. van Eikema Hommes, Ashish Radadiya, Jochen Heil, Steven A. Benner, Timothy Clark, Stefan M. Kast, Nigel G. J. Richards. Tautomeric Equilibria of Nucleobases in the Hachimoji Expanded Genetic Alphabet. Journal of Chemical Theory and Computation 2020, 16 (4) , 2766-2777. https://doi.org/10.1021/acs.jctc.9b01079
  4. Vivian T. Dien, Matthew Holcomb, Aaron W. Feldman, Emil C. Fischer, Tammy J. Dwyer, Floyd E. Romesberg. Progress Toward a Semi-Synthetic Organism with an Unrestricted Expanded Genetic Alphabet. Journal of the American Chemical Society 2018, 140 (47) , 16115-16123. https://doi.org/10.1021/jacs.8b08416
  5. Aaron W. Feldman and Floyd E. Romesberg . Expansion of the Genetic Alphabet: A Chemist’s Approach to Synthetic Biology. Accounts of Chemical Research 2018, 51 (2) , 394-403. https://doi.org/10.1021/acs.accounts.7b00403
  6. Michael P. Ledbetter, Rebekah J. Karadeema, and Floyd E. Romesberg . Reprograming the Replisome of a Semisynthetic Organism for the Expansion of the Genetic Alphabet. Journal of the American Chemical Society 2018, 140 (2) , 758-765. https://doi.org/10.1021/jacs.7b11488
  7. Qian Wang, Xiao-Ying Xie, Juan Han, and Ganglong Cui . QM and QM/MM Studies on Excited-State Relaxation Mechanisms of Unnatural Bases in Vacuo and Base Pairs in DNA. The Journal of Physical Chemistry B 2017, 121 (46) , 10467-10478. https://doi.org/10.1021/acs.jpcb.7b09046
  8. Sydney E. Morris, Aaron W. Feldman, and Floyd E. Romesberg . Synthetic Biology Parts for the Storage of Increased Genetic Information in Cells. ACS Synthetic Biology 2017, 6 (10) , 1834-1840. https://doi.org/10.1021/acssynbio.7b00115
  9. Michiko Kimoto and Ichiro Hirao . Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in Double-Stranded DNAs. ACS Synthetic Biology 2017, 6 (10) , 1944-1951. https://doi.org/10.1021/acssynbio.7b00165
  10. Aaron W. Feldman and Floyd E. Romesberg . In Vivo Structure–Activity Relationships and Optimization of an Unnatural Base Pair for Replication in a Semi-Synthetic Organism. Journal of the American Chemical Society 2017, 139 (33) , 11427-11433. https://doi.org/10.1021/jacs.7b03540
  11. Aaron W. Feldman, Vivian T. Dien, and Floyd E. Romesberg . Chemical Stabilization of Unnatural Nucleotide Triphosphates for the in Vivo Expansion of the Genetic Alphabet. Journal of the American Chemical Society 2017, 139 (6) , 2464-2467. https://doi.org/10.1021/jacs.6b12731
  12. Liqin Zhang, Zunyi Yang, Kwame Sefah, Kevin M. Bradley, Shuichi Hoshika, Myong-Jung Kim, Hyo-Joong Kim, Guizhi Zhu, Elizabeth Jiménez, Sena Cansiz, I-Ting Teng, Carole Champanhac, Christopher McLendon, Chen Liu, Wen Zhang, Dietlind L. Gerloff, Zhen Huang, Weihong Tan, and Steven A. Benner . Evolution of Functional Six-Nucleotide DNA. Journal of the American Chemical Society 2015, 137 (21) , 6734-6737. https://doi.org/10.1021/jacs.5b02251
  13. Millie M. Georgiadis, Isha Singh, Whitney F. Kellett, Shuichi Hoshika, Steven A. Benner, and Nigel G. J. Richards . Structural Basis for a Six Nucleotide Genetic Alphabet. Journal of the American Chemical Society 2015, 137 (21) , 6947-6955. https://doi.org/10.1021/jacs.5b03482
  14. Sk Jahiruddin and Ayan Datta . What Sustains the Unnatural Base Pairs (UBPs) with No Hydrogen Bonds. The Journal of Physical Chemistry B 2015, 119 (18) , 5839-5845. https://doi.org/10.1021/acs.jpcb.5b03293
  15. Shuangluo Xia and William H. Konigsberg . RB69 DNA Polymerase Structure, Kinetics, and Fidelity. Biochemistry 2014, 53 (17) , 2752-2767. https://doi.org/10.1021/bi4014215
  16. Floyd E. Romesberg. Creation, Optimization, and Use of Semi-Synthetic Organisms that Store and Retrieve Increased Genetic Information. Journal of Molecular Biology 2022, 434 (8) , 167331. https://doi.org/10.1016/j.jmb.2021.167331
  17. N. R. Jena, P. Das. Hybrid nucleobases as new and efficient unnatural genetic letters. Journal of Biomolecular Structure and Dynamics 2021, 23 , 1-11. https://doi.org/10.1080/07391102.2021.2003863
  18. Juntaek Oh, Ji Shin, Ilona Christy Unarta, Wei Wang, Aaron W. Feldman, Rebekah J. Karadeema, Liang Xu, Jun Xu, Jenny Chong, Ramanarayanan Krishnamurthy, Xuhui Huang, Floyd E. Romesberg, Dong Wang. Transcriptional processing of an unnatural base pair by eukaryotic RNA polymerase II. Nature Chemical Biology 2021, 17 (8) , 906-914. https://doi.org/10.1038/s41589-021-00817-3
  19. Paola Handal-Marquez, Anupama Anupama, Valerie Pezo, Philippe Marlière, Piet Herdewijn, Vitor B. Pinheiro. Beneath the XNA world: Tools and targets to build novel biology. Current Opinion in Systems Biology 2020, 24 , 142-152. https://doi.org/10.1016/j.coisb.2020.10.013
  20. Zahra Ouaray, Steven A. Benner, Millie M. Georgiadis, Nigel G.J. Richards. Building better polymerases: Engineering the replication of expanded genetic alphabets. Journal of Biological Chemistry 2020, 295 (50) , 17046-17059. https://doi.org/10.1074/jbc.REV120.013745
  21. Michiko Kimoto, Ichiro Hirao. Genetic alphabet expansion technology by creating unnatural base pairs. Chemical Society Reviews 2020, 49 (21) , 7602-7626. https://doi.org/10.1039/D0CS00457J
  22. Rodrigo Galindo-Murillo, Joaquín Barroso-Flores. Hydrophobic unnatural base pairs show a Watson-Crick pairing in micro-second molecular dynamics simulations. Journal of Biomolecular Structure and Dynamics 2020, 38 (14) , 4098-4106. https://doi.org/10.1080/07391102.2019.1671898
  23. Emil C. Fischer, Koji Hashimoto, Yorke Zhang, Aaron W. Feldman, Vivian T. Dien, Rebekah J. Karadeema, Ramkrishna Adhikary, Michael P. Ledbetter, Ramanarayanan Krishnamurthy, Floyd E. Romesberg. New codons for efficient production of unnatural proteins in a semisynthetic organism. Nature Chemical Biology 2020, 16 (5) , 570-576. https://doi.org/10.1038/s41589-020-0507-z
  24. Andreas Marx, Karin Betz. The Structural Basis for Processing of Unnatural Base Pairs by DNA Polymerases. Chemistry – A European Journal 2020, 26 (16) , 3446-3463. https://doi.org/10.1002/chem.201903525
  25. Ali Nikoomanzar, Nicholas Chim, Eric J. Yik, John C. Chaput. Engineering polymerases for applications in synthetic biology. Quarterly Reviews of Biophysics 2020, 53 https://doi.org/10.1017/S0033583520000050
  26. Huiqing Zhou, Simone Rauch, Qing Dai, Xiaolong Cui, Zijie Zhang, Sigrid Nachtergaele, Caraline Sepich, Chuan He, Bryan C. Dickinson. Evolution of a reverse transcriptase to map N1-methyladenosine in human messenger RNA. Nature Methods 2019, 16 (12) , 1281-1288. https://doi.org/10.1038/s41592-019-0550-4
  27. Heike Maria Kropp, Simon Leonard Dürr, Christine Peter, Kay Diederichs, Andreas Marx. Snapshots of a modified nucleotide moving through the confines of a DNA polymerase. Proceedings of the National Academy of Sciences 2018, 115 (40) , 9992-9997. https://doi.org/10.1073/pnas.1811518115
  28. Vivian T Dien, Sydney E Morris, Rebekah J Karadeema, Floyd E Romesberg. Expansion of the genetic code via expansion of the genetic alphabet. Current Opinion in Chemical Biology 2018, 46 , 196-202. https://doi.org/10.1016/j.cbpa.2018.08.009
  29. Isha Singh, Roberto Laos, Shuichi Hoshika, Steven A Benner, Millie M Georgiadis. Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide. Nucleic Acids Research 2018, 46 (15) , 7977-7988. https://doi.org/10.1093/nar/gky552
  30. Kyung Hyun Lee, Kiyofumi Hamashima, Michiko Kimoto, Ichiro Hirao. Genetic alphabet expansion biotechnology by creating unnatural base pairs. Current Opinion in Biotechnology 2018, 51 , 8-15. https://doi.org/10.1016/j.copbio.2017.09.006
  31. Wei-Wei Guo, Teng-Shuo Zhang, Wei-Hai Fang, Ganglong Cui. QM/MM studies on the excited-state relaxation mechanism of a semisynthetic dTPT3 base. Physical Chemistry Chemical Physics 2018, 20 (7) , 5067-5073. https://doi.org/10.1039/C7CP08696B
  32. Rakesh K. Tiwari, Rajendra P. Ojha, Gargi Tiwari, Vishnudatt Pandey, Vijaysree Mall. Molecular dynamics study of some non-hydrogen-bonding base pair DNA strands. 2018,,, 130030. https://doi.org/10.1063/1.5033174
  33. Kaitlin M Pugliese, Gregory A Weiss. Recent progress in dissecting molecular recognition by DNA polymerases with non-native substrates. Current Opinion in Chemical Biology 2017, 41 , 43-49. https://doi.org/10.1016/j.cbpa.2017.10.005
  34. Steven Pauff, Jamie M Withers, Iain JW McKean, Simon P Mackay, Glenn A Burley. Synthetic biological approaches for RNA labelling and imaging: design principles and future opportunities. Current Opinion in Biotechnology 2017, 48 , 153-158. https://doi.org/10.1016/j.copbio.2017.04.003
  35. Karin Betz, Michiko Kimoto, Kay Diederichs, Ichiro Hirao, Andreas Marx. Strukturelle Studie zur Erweiterung des genetischen Codes durch ein artifizielles Nucleobasenpaar. Angewandte Chemie 2017, 129 (39) , 12162-12166. https://doi.org/10.1002/ange.201704190
  36. Karin Betz, Michiko Kimoto, Kay Diederichs, Ichiro Hirao, Andreas Marx. Structural Basis for Expansion of the Genetic Alphabet with an Artificial Nucleobase Pair. Angewandte Chemie International Edition 2017, 56 (39) , 12000-12003. https://doi.org/10.1002/anie.201704190
  37. Najeh Rekik, Majid F. Alshammari. Electrical anharmonicity and dampings contributions to Cl- H → stretching band in gaseous (CH 3 ) 2 O…HCl complex: Quantum dynamic study and prediction of the temperature effects. Chemical Physics Letters 2017, 678 , 222-232. https://doi.org/10.1016/j.cplett.2017.04.054
  38. Fuyi Zhang, Yan Liang, Jing Li, Fei Gao, Hong Liu, Yufen Zhao. A Concise Synthesis of Novel Aryl Pyrimidine C -Nucleoside Analogs from Sugar Alkynes. Asian Journal of Organic Chemistry 2017, 6 (5) , 561-565. https://doi.org/10.1002/ajoc.201600583
  39. Robert W. Molt, Millie M. Georgiadis, Nigel G.J. Richards. Consecutive non-natural PZ nucleobase pairs in DNA impact helical structure as seen in 50 μs molecular dynamics simulations. Nucleic Acids Research 2017, 45 (7) , 3643-3653. https://doi.org/10.1093/nar/gkx144
  40. Audrey Hottin, Karin Betz, Kay Diederichs, Andreas Marx. Structural Basis for the KlenTaq DNA Polymerase Catalysed Incorporation of Alkene- versus Alkyne-Modified Nucleotides. Chemistry - A European Journal 2017, 23 (9) , 2109-2118. https://doi.org/10.1002/chem.201604515
  41. Yorke Zhang, Brian M. Lamb, Aaron W. Feldman, Anne Xiaozhou Zhou, Thomas Lavergne, Lingjun Li, Floyd E. Romesberg. A semisynthetic organism engineered for the stable expansion of the genetic alphabet. Proceedings of the National Academy of Sciences 2017, 114 (6) , 1317-1322. https://doi.org/10.1073/pnas.1616443114
  42. Falk Wachowius, James Attwater, Philipp Holliger. Nucleic acids: function and potential for abiogenesis. Quarterly Reviews of Biophysics 2017, 50 https://doi.org/10.1017/S0033583517000038
  43. Rodrigo Galindo-Murillo, Joaquín Barroso-Flores. Structural and dynamical instability of DNA caused by high occurrence of d5SICS and dNaM unnatural nucleotides. Physical Chemistry Chemical Physics 2017, 19 (16) , 10571-10580. https://doi.org/10.1039/C7CP01477E
  44. Indu Negi, Preetleen Kathuria, Purshotam Sharma, Stacey D. Wetmore. How do hydrophobic nucleobases differ from natural DNA nucleobases? Comparison of structural features and duplex properties from QM calculations and MD simulations. Physical Chemistry Chemical Physics 2017, 19 (25) , 16365-16374. https://doi.org/10.1039/C7CP02576A
  45. Brennan Ashwood, Marvin Pollum, Carlos E. Crespo-Hernández. Can a Six-Letter Alphabet Increase the Likelihood of Photochemical Assault to the Genetic Code?. Chemistry - A European Journal 2016, 22 (46) , 16648-16656. https://doi.org/10.1002/chem.201602160
  46. Markus Probst, Walter Aeschimann, Thi T.H. Chau, Simon M. Langenegger, Achim Stocker, Robert Häner. Structural insight into DNA-assembled oligochromophores: crystallographic analysis of pyrene- and phenanthrene-modified DNA in complex with BpuJI endonuclease. Nucleic Acids Research 2016, 44 (15) , 7079-7089. https://doi.org/10.1093/nar/gkw644
  47. Ralph P. G. Bosmans, Jeroen M. Briels, Lech-Gustav Milroy, Tom F. A. de Greef, Maarten Merkx, Luc Brunsveld. Supramolecular Control over Split-Luciferase Complementation. Angewandte Chemie 2016, 128 (31) , 9045-9049. https://doi.org/10.1002/ange.201602807
  48. Ralph P. G. Bosmans, Jeroen M. Briels, Lech-Gustav Milroy, Tom F. A. de Greef, Maarten Merkx, Luc Brunsveld. Supramolecular Control over Split-Luciferase Complementation. Angewandte Chemie International Edition 2016, 55 (31) , 8899-8903. https://doi.org/10.1002/anie.201602807
  49. Stephanie Kath-Schorr. Cycloadditions for Studying Nucleic Acids. Topics in Current Chemistry 2016, 374 (1) https://doi.org/10.1007/s41061-015-0004-0
  50. Wenjuan Wang, Xiehuang Sheng, Shaolong Zhang, Fang Huang, Chuanzhi Sun, Jianbiao Liu, Dezhan Chen. Theoretical characterization of the conformational features of unnatural oligonucleotides containing a six nucleotide genetic alphabet. Physical Chemistry Chemical Physics 2016, 18 (41) , 28492-28501. https://doi.org/10.1039/C6CP05594J
  51. Subhendu Sekhar Bag, Sangita Talukdar, Suman Kalyan Das, Manoj Kumar Pradhan, Soumen Mukherjee. Donor/acceptor chromophores-decorated triazolyl unnatural nucleosides: synthesis, photophysical properties and study of interaction with BSA. Organic & Biomolecular Chemistry 2016, 14 (22) , 5088-5108. https://doi.org/10.1039/C6OB00500D
  52. Anna Tokarenko, Lenka Poštová Slavětínská, Blanka Klepetářová, Michal Hocek. Synthesis of Benzene and Pyridine 2′- C -Methyl- C -ribonucleosides and -nucleotides. European Journal of Organic Chemistry 2015, 2015 (36) , 7962-7983. https://doi.org/10.1002/ejoc.201501219
  53. Steven K. Wolk, Richard K. Shoemaker, Wes S. Mayfield, Andrew L. Mestdagh, Nebojsa Janjic. Influence of 5-N-carboxamide modifications on the thermodynamic stability of oligonucleotides. Nucleic Acids Research 2015, 43 (19) , 9107-9122. https://doi.org/10.1093/nar/gkv981
  54. Denis A. Malyshev, Floyd E. Romesberg. Das erweiterte genetische Alphabet. Angewandte Chemie 2015, 127 (41) , 12098-12113. https://doi.org/10.1002/ange.201502890
  55. Denis A. Malyshev, Floyd E. Romesberg. The Expanded Genetic Alphabet. Angewandte Chemie International Edition 2015, 54 (41) , 11930-11944. https://doi.org/10.1002/anie.201502890
  56. Armando R. Hernandez, Yaming Shao, Shuichi Hoshika, Zunyi Yang, Sandip A. Shelke, Julien Herrou, Hyo-Joong Kim, Myong-Jung Kim, Joseph A. Piccirilli, Steven A. Benner. A Crystal Structure of a Functional RNA Molecule Containing an Artificial Nucleobase Pair. Angewandte Chemie 2015, 127 (34) , 9991-9994. https://doi.org/10.1002/ange.201504731
  57. Armando R. Hernandez, Yaming Shao, Shuichi Hoshika, Zunyi Yang, Sandip A. Shelke, Julien Herrou, Hyo‐Joong Kim, Myong‐Jung Kim, Joseph A. Piccirilli, Steven A. Benner. A Crystal Structure of a Functional RNA Molecule Containing an Artificial Nucleobase Pair. Angewandte Chemie International Edition 2015, 54 (34) , 9853-9856. https://doi.org/10.1002/anie.201504731
  58. Noriko Tarashima, Yasuo Komatsu, Kazuhiro Furukawa, Noriaki Minakawa. Faithful PCR Amplification of an Unnatural Base-Pair Analogue with Four Hydrogen Bonds. Chemistry - A European Journal 2015, 21 (30) , 10688-10695. https://doi.org/10.1002/chem.201501484
  59. Christof Domnick, Frank Eggert, Stephanie Kath-Schorr. Site-specific enzymatic introduction of a norbornene modified unnatural base into RNA and application in post-transcriptional labeling. Chemical Communications 2015, 51 (39) , 8253-8256. https://doi.org/10.1039/C5CC01765C
  60. Meidong Zhu, Chao Luo, Fangwei Zhang, Fei Liu, Jingyan Zhang, Shouwu Guo. Interactions of the primers and Mg 2+ with graphene quantum dots enhance PCR performance. RSC Advances 2015, 5 (91) , 74515-74522. https://doi.org/10.1039/C5RA12729G
  61. Márcia K. D. L. Belarmino, Vanessa F. Cruz, Nathália B. D. Lima. Hydrogen-bonded complexes between dimethyl sulfoxide and monoprotic acids: molecular properties and IR spectroscopy. Journal of Molecular Modeling 2014, 20 (11) https://doi.org/10.1007/s00894-014-2477-0
  62. Kirandeep Dhami, Denis A. Malyshev, Phillip Ordoukhanian, Tomáš Kubelka, Michal Hocek, Floyd E. Romesberg. Systematic exploration of a class of hydrophobic unnatural base pairs yields multiple new candidates for the expansion of the genetic alphabet. Nucleic Acids Research 2014, 42 (16) , 10235-10244. https://doi.org/10.1093/nar/gku715
  63. Denis A. Malyshev, Kirandeep Dhami, Thomas Lavergne, Tingjian Chen, Nan Dai, Jeremy M. Foster, Ivan R. Corrêa, Floyd E. Romesberg. A semi-synthetic organism with an expanded genetic alphabet. Nature 2014, 509 (7500) , 385-388. https://doi.org/10.1038/nature13314

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

MENDELEY PAIRING EXPIRED
Your Mendeley pairing has expired. Please reconnect

This website uses cookies to improve your user experience. By continuing to use the site, you are accepting our use of cookies. Read the ACS privacy policy.

CONTINUE