Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Mar 15;25(6):1272–1280. doi: 10.1093/nar/25.6.1272

Synthesis and studies on the effect of 2-thiouridine and 4-thiouridine on sugar conformation and RNA duplex stability.

R K Kumar 1, D R Davis 1
PMCID: PMC146581  PMID: 9092639

Abstract

In order to understand the effect of 2-thiouridine (s2U) substitution on RNA structure and the potential for stabilization of tRNA codon-anticodon interactions through s2U-34 modification, a pentamer RNA sequence, Gs2UUUC, was synthesized and characterized by NMR spectroscopy. The single strand contains the UUU anticodon sequence of tRNALys with flanking GCs to increase duplex stability. Regiochemical effects of uridine thiolation were determined by comparing the structure and stability of the 2-thiouridine containing oligonucleotide with an identical sequence containing 4-thiouridine (s4U) and also the normal uridine nucleoside. Circular dichroism spectrum indicated an A-form helical conformation for Gs2UUUC which was further confirmed by 2D ROESY NMR experiments. The duplex stability of the three pentamers complexed with a 2'-O-methyl-ribonucleotide complementary strand, GmAmAmAmCm, was determined by UV thermal melting studies and by 1H NMR spectroscopy. The duplex containing s2U has a T m of 30.7 degrees C compared to 19. 0 degrees C for the unmodified control and 14.5 degrees C for the s4U containing duplex. The results from UV experiments were corroborated by imino proton NMR studies that show proton exchange rates, chemical shift differences, and NH proton linewidths indicative of the stability order s2U >U >s4U. The magnitude of the effect of s2U in our model system is comparable to the 20 degrees C stabilization observed by Grosjean and co-workers for 2-thiolation in a codon-anticodon model system composed of two tRNAs with complementary anticodon sequences [Houssier, C., Degee, P., Nicoghosian, K. and Grosjean, H. (1988) J. Biomol. Struct. Dyn., 5, 1259-1266].

Full Text

The Full Text of this article is available as a PDF (185.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agris P. F., Brown S. C. Systems for the NMR study of modified nucleoside-dependent, metal-ion induced conformational changes in nucleic acids. Methods Enzymol. 1995;261:270–299. doi: 10.1016/s0076-6879(95)61014-6. [DOI] [PubMed] [Google Scholar]
  2. Agris P. F., Malkiewicz A., Kraszewski A., Everett K., Nawrot B., Sochacka E., Jankowska J., Guenther R. Site-selected introduction of modified purine and pyrimidine ribonucleosides into RNA by automated phosphoramidite chemistry. Biochimie. 1995;77(1-2):125–134. doi: 10.1016/0300-9084(96)88115-6. [DOI] [PubMed] [Google Scholar]
  3. Agris P. F. The importance of being modified: roles of modified nucleosides and Mg2+ in RNA structure and function. Prog Nucleic Acid Res Mol Biol. 1996;53:79–129. doi: 10.1016/s0079-6603(08)60143-9. [DOI] [PubMed] [Google Scholar]
  4. Agris P. F. Wobble position modified nucleosides evolved to select transfer RNA codon recognition: a modified-wobble hypothesis. Biochimie. 1991 Nov;73(11):1345–1349. doi: 10.1016/0300-9084(91)90163-u. [DOI] [PubMed] [Google Scholar]
  5. Altona C., Sundaralingam M. Conformational analysis of the sugar ring in nucleosides and nucleotides. Improved method for the interpretation of proton magnetic resonance coupling constants. J Am Chem Soc. 1973 Apr 4;95(7):2333–2344. doi: 10.1021/ja00788a038. [DOI] [PubMed] [Google Scholar]
  6. Blommers M. J., Pieles U., De Mesmaeker A. An approach to the structure determination of nucleic acid analogues hybridized to RNA. NMR studies of a duplex between 2'-OMe RNA and an oligonucleotide containing a single amide backbone modification. Nucleic Acids Res. 1994 Oct 11;22(20):4187–4194. doi: 10.1093/nar/22.20.4187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Damha M. J., Ogilvie K. K. Oligoribonucleotide synthesis. The silyl-phosphoramidite method. Methods Mol Biol. 1993;20:81–114. doi: 10.1385/0-89603-281-7:81. [DOI] [PubMed] [Google Scholar]
  8. Davanloo P., Sprinzl M., Watanabe K., Albani M., Kersten H. Role of ribothymidine in the thermal stability of transfer RNA as monitored by proton magnetic resonance. Nucleic Acids Res. 1979 Apr;6(4):1571–1581. doi: 10.1093/nar/6.4.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gasparutto D., Livache T., Bazin H., Duplaa A. M., Guy A., Khorlin A., Molko D., Roget A., Téoule R. Chemical synthesis of a biologically active natural tRNA with its minor bases. Nucleic Acids Res. 1992 Oct 11;20(19):5159–5166. doi: 10.1093/nar/20.19.5159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Griffey R. H., Davis D. R., Yamaizumi Z., Nishimura S., Hawkins B. L., Poulter C. D. 15N-labeled tRNA. Identification of 4-thiouridine in Escherichia coli tRNASer1 and tRNATyr2 by 1H-15N two-dimensional NMR spectroscopy. J Biol Chem. 1986 Sep 15;261(26):12074–12078. [PubMed] [Google Scholar]
  11. Hall K. B., McLaughlin L. W. Thermodynamic and structural properties of pentamer DNA.DNA, RNA.RNA, and DNA.RNA duplexes of identical sequence. Biochemistry. 1991 Nov 5;30(44):10606–10613. doi: 10.1021/bi00108a002. [DOI] [PubMed] [Google Scholar]
  12. Houssier C., Degée P., Nicoghosian K., Grosjean H. Effect of uridine dethiolation in the anticodon triplet of tRNA(Glu) on its association with tRNA(Phe). J Biomol Struct Dyn. 1988 Jun;5(6):1259–1266. doi: 10.1080/07391102.1988.10506468. [DOI] [PubMed] [Google Scholar]
  13. Inoue H., Hayase Y., Imura A., Iwai S., Miura K., Ohtsuka E. Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides. Nucleic Acids Res. 1987 Aug 11;15(15):6131–6148. doi: 10.1093/nar/15.15.6131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kawai G., Yamamoto Y., Kamimura T., Masegi T., Sekine M., Hata T., Iimori T., Watanabe T., Miyazawa T., Yokoyama S. Conformational rigidity of specific pyrimidine residues in tRNA arises from posttranscriptional modifications that enhance steric interaction between the base and the 2'-hydroxyl group. Biochemistry. 1992 Feb 4;31(4):1040–1046. doi: 10.1021/bi00119a012. [DOI] [PubMed] [Google Scholar]
  15. Khare D., Orban J. Synthesis of backbone deuterium labelled [r(CGCGAAUUCGCG)]2 and HPLC purification of synthetic RNA. Nucleic Acids Res. 1992 Oct 11;20(19):5131–5136. doi: 10.1093/nar/20.19.5131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laing L. G., Draper D. E. Thermodynamics of RNA folding in a conserved ribosomal RNA domain. J Mol Biol. 1994 Apr 15;237(5):560–576. doi: 10.1006/jmbi.1994.1255. [DOI] [PubMed] [Google Scholar]
  17. Lee C. H., Tinoco I., Jr Conformation studies of 13 trinucleoside diphosphates by 360 MHz PMR spectroscopy. A bulged base conformation. I. Base protons and H1' protons. Biophys Chem. 1980 Apr;11(2):283–294. doi: 10.1016/0301-4622(80)80031-7. [DOI] [PubMed] [Google Scholar]
  18. Lee C. H., Tinoco I., Jr Studies of the conformation of modified dinucleoside phosphates containing 1,N6-ethenoadenosine and 2'-O-methylcytidine by 360-MHz 1H nuclear magnetic resonance spectroscopy. Investigation of the solution conformations of dinucleoside phosphates. Biochemistry. 1977 Dec 13;16(25):5403–5414. doi: 10.1021/bi00644a001. [DOI] [PubMed] [Google Scholar]
  19. Lesnik E. A., Guinosso C. J., Kawasaki A. M., Sasmor H., Zounes M., Cummins L. L., Ecker D. J., Cook P. D., Freier S. M. Oligodeoxynucleotides containing 2'-O-modified adenosine: synthesis and effects on stability of DNA:RNA duplexes. Biochemistry. 1993 Aug 3;32(30):7832–7838. doi: 10.1021/bi00081a031. [DOI] [PubMed] [Google Scholar]
  20. Limbach P. A., Crain P. F., McCloskey J. A. Summary: the modified nucleosides of RNA. Nucleic Acids Res. 1994 Jun 25;22(12):2183–2196. doi: 10.1093/nar/22.12.2183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mazumdar S. K., Saenger W. Molecular structure of poly-2-thiouridylic acid, a double helix with non-equivalent polynucleotide chains. J Mol Biol. 1974 May 15;85(2):213–219. doi: 10.1016/0022-2836(74)90361-1. [DOI] [PubMed] [Google Scholar]
  22. Ni J., Pomerantz C., Rozenski J., Zhang Y., McCloskey J. A. Interpretation of oligonucleotide mass spectra for determination of sequence using electrospray ionization and tandem mass spectrometry. Anal Chem. 1996 Jul 1;68(13):1989–1999. doi: 10.1021/ac960270t. [DOI] [PubMed] [Google Scholar]
  23. Pomerantz S. C., McCloskey J. A. Analysis of RNA hydrolyzates by liquid chromatography-mass spectrometry. Methods Enzymol. 1990;193:796–824. doi: 10.1016/0076-6879(90)93452-q. [DOI] [PubMed] [Google Scholar]
  24. Shah K., Wu H., Rana T. M. Synthesis of uridine phosphoramidite analogs: reagents for site-specific incorporation of photoreactive sites into RNA sequences. Bioconjug Chem. 1994 Nov-Dec;5(6):508–512. doi: 10.1021/bc00030a005. [DOI] [PubMed] [Google Scholar]
  25. Steinberg S., Misch A., Sprinzl M. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1993 Jul 1;21(13):3011–3015. doi: 10.1093/nar/21.13.3011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sugimoto N., Nakano S., Katoh M., Matsumura A., Nakamuta H., Ohmichi T., Yoneyama M., Sasaki M. Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry. 1995 Sep 5;34(35):11211–11216. doi: 10.1021/bi00035a029. [DOI] [PubMed] [Google Scholar]
  27. Varani G., Tinoco I., Jr RNA structure and NMR spectroscopy. Q Rev Biophys. 1991 Nov;24(4):479–532. doi: 10.1017/s0033583500003875. [DOI] [PubMed] [Google Scholar]
  28. Watanabe K., Hayashi N., Oyama A., Nishikawa K., Ueda T., Miura K. Unusual anticodon loop structure found in E.coli lysine tRNA. Nucleic Acids Res. 1994 Jan 11;22(1):79–87. doi: 10.1093/nar/22.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Watanabe K., Oshima T., Nishimura S. CD spectra of 5-methyl-2-thiouridine in tRNA-Met-f from an extreme thermophile. Nucleic Acids Res. 1976 Jul;3(7):1703–1713. doi: 10.1093/nar/3.7.1703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Watanabe K. Reactions of 2-thioribothymidine ad 4-thiouridine with hydrogen peroxide in transfer ribonucleic acids from Thermus thermophilus and Escherichia coli as studied by circular dichroism. Biochemistry. 1980 Nov 25;19(24):5542–5549. doi: 10.1021/bi00565a013. [DOI] [PubMed] [Google Scholar]
  31. Watanabe K., Yokoyama S., Hansske F., Kasai H., Miyazawa T. CD and NMR studies on the conformational thermostability of 2-thioribothymidine found in the T psi C loop of thermophile tRNA. Biochem Biophys Res Commun. 1979 Nov 28;91(2):671–677. doi: 10.1016/0006-291x(79)91574-2. [DOI] [PubMed] [Google Scholar]
  32. Westman E., Strömberg R. Removal of t-butyldimethylsilyl protection in RNA-synthesis. Triethylamine trihydrofluoride (TEA, 3HF) is a more reliable alternative to tetrabutylammonium fluoride (TBAF). Nucleic Acids Res. 1994 Jun 25;22(12):2430–2431. doi: 10.1093/nar/22.12.2430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wincott F., DiRenzo A., Shaffer C., Grimm S., Tracz D., Workman C., Sweedler D., Gonzalez C., Scaringe S., Usman N. Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res. 1995 Jul 25;23(14):2677–2684. doi: 10.1093/nar/23.14.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yamamoto Y., Yokoyama S., Miyazawa T., Watanabe K., Higuchi S. NMR analyses on the molecular mechanism of the conformational rigidity of 2-thioribothymidine, a modified nucleoside in extreme thermophile tRNAs. FEBS Lett. 1983 Jun 27;157(1):95–99. doi: 10.1016/0014-5793(83)81123-5. [DOI] [PubMed] [Google Scholar]
  35. Yokoyama S., Watanabe T., Murao K., Ishikura H., Yamaizumi Z., Nishimura S., Miyazawa T. Molecular mechanism of codon recognition by tRNA species with modified uridine in the first position of the anticodon. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4905–4909. doi: 10.1073/pnas.82.15.4905. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES