Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1995 Oct 11;23(19):3916–3921. doi: 10.1093/nar/23.19.3916

Effects of site-specific substitution of 5-fluorouridine on the stabilities of duplex DNA and RNA.

P V Sahasrabudhe 1, R T Pon 1, W H Gmeiner 1
PMCID: PMC307310  PMID: 7479036

Abstract

The effects of 5-fluorouridine (FUrd) and 5-fluorodeoxyuridine (FdUrd) substitution on the stabilities of duplex RNA and DNA have been studied to determine how FUrd substitution in nucleic acids may alter the efficiency of biochemical processes that require complementary base pairing for molecular recognition. The parent sequence, 5'-GCGAAUUCGC, contains two non-equivalent uridines. Eight oligonucleotides (four RNA and four DNA) were prepared with either zero, one or two Urd substituted by FUrd. The stability of each self-complementary duplex was determined by measuring the absorbance at 260 nm as a function of temperature. Tm values were calculated from the first derivative of the absorbance versus temperature profiles and values for delta H0 and delta S0 were calculated from the concentration dependence of the Tm. Individual absorbance versus temperature curves were also analyzed by a parametric approach to calculate thermodynamic parameters for the duplex to single-stranded transition. Analysis of the thermodynamic parameters for each oligonucleotide revealed that FUrd substitution had sequence-dependent effects in both A-form RNA and B-form DNA duplexes. Conservation of helix geometry in FUrd-substituted duplexes was determined by CD spectroscopy. FUrd substitution at a single site in RNA stabilized the duplex (delta delta G37 = 0.8 kcal/mol), largely due to more favorable stacking interactions. FdUrd substitution at a single site in DNA destabilized the duplex (delta delta G37 = 0.3 kcal/mol) as a consequence of less favorable stacking interactions. All duplexes melt via single cooperative transitions.

Full text

PDF
3916

Selected References

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

  1. Armstrong R. D., Takimoto C. H., Cadman E. C. Fluoropyrimidine-mediated changes in small nuclear RNA. J Biol Chem. 1986 Jan 5;261(1):21–24. [PubMed] [Google Scholar]
  2. Borer P. N., Dengler B., Tinoco I., Jr, Uhlenbeck O. C. Stability of ribonucleic acid double-stranded helices. J Mol Biol. 1974 Jul 15;86(4):843–853. doi: 10.1016/0022-2836(74)90357-x. [DOI] [PubMed] [Google Scholar]
  3. Breslauer K. J., Sturtevant J. M. A calorimetric investigation of single stranded base stacking in the ribo-oligonucleotide A7. Biophys Chem. 1977 Nov;7(3):205–209. doi: 10.1016/0301-4622(77)87023-3. [DOI] [PubMed] [Google Scholar]
  4. Daher G. C., Harris B. E., Diasio R. B. Metabolism of pyrimidine analogues and their nucleosides. Pharmacol Ther. 1990;48(2):189–222. doi: 10.1016/0163-7258(90)90080-l. [DOI] [PubMed] [Google Scholar]
  5. Dolnick B. J., Pink J. J. Effects of 5-fluorouracil on dihydrofolate reductase and dihydrofolate reductase mRNA from methotrexate-resistant KB cells. J Biol Chem. 1985 Mar 10;260(5):3006–3014. [PubMed] [Google Scholar]
  6. Freier S. M., Hill K. O., Dewey T. G., Marky L. A., Breslauer K. J., Turner D. H. Solvent effects on the kinetics and thermodynamics of stacking in poly(cytidylic acid). Biochemistry. 1981 Mar 17;20(6):1419–1426. doi: 10.1021/bi00509a003. [DOI] [PubMed] [Google Scholar]
  7. Gollnick P., Hardin C. C., Horowitz J. Fluorine-19 nuclear magnetic resonance study of codon-anticodon interaction in 5-fluorouracil-substituted E. coli transfer RNAs. Nucleic Acids Res. 1986 Jun 11;14(11):4659–4672. doi: 10.1093/nar/14.11.4659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Guenther R. H., Hardin C. C., Sierzputowska-Gracz H., Dao V., Agris P. F. A magnesium-induced conformational transition in the loop of a DNA analog of the yeast tRNA(Phe) anticodon is dependent on RNA-like modifications of the bases of the stem. Biochemistry. 1992 Nov 17;31(45):11004–11011. doi: 10.1021/bi00160a009. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Kealey J. T., Santi D. V. Stereochemistry of tRNA(m5U54)-methyltransferase catalysis: 19F NMR spectroscopy of an enzyme-FUraRNA covalent complex. Biochemistry. 1995 Feb 28;34(8):2441–2446. doi: 10.1021/bi00008a006. [DOI] [PubMed] [Google Scholar]
  11. Kremer A. B., Mikita T., Beardsley G. P. Chemical consequences of incorporation of 5-fluorouracil into DNA as studied by NMR. Biochemistry. 1987 Jan 27;26(2):391–397. doi: 10.1021/bi00376a009. [DOI] [PubMed] [Google Scholar]
  12. LeBlanc D. A., Morden K. M. Thermodynamic characterization of deoxyribooligonucleotide duplexes containing bulges. Biochemistry. 1991 Apr 23;30(16):4042–4047. doi: 10.1021/bi00230a031. [DOI] [PubMed] [Google Scholar]
  13. Lenz H. J., Manno D. J., Danenberg K. D., Danenberg P. V. Incorporation of 5-fluorouracil into U2 and U6 snRNA inhibits mRNA precursor splicing. J Biol Chem. 1994 Dec 16;269(50):31962–31968. [PubMed] [Google Scholar]
  14. Martin F. H., Tinoco I., Jr DNA-RNA hybrid duplexes containing oligo(dA:rU) sequences are exceptionally unstable and may facilitate termination of transcription. Nucleic Acids Res. 1980 May 24;8(10):2295–2299. doi: 10.1093/nar/8.10.2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nelson J. W., Tinoco I., Jr Comparison of the kinetics of ribooligonucleotide, deoxyribooligonucleotide, and hybrid oligonucleotide double-strand formation by temperature-jump kinetics. Biochemistry. 1982 Oct 12;21(21):5289–5295. doi: 10.1021/bi00264a026. [DOI] [PubMed] [Google Scholar]
  16. Parker W. B., Cheng Y. C. Metabolism and mechanism of action of 5-fluorouracil. Pharmacol Ther. 1990;48(3):381–395. doi: 10.1016/0163-7258(90)90056-8. [DOI] [PubMed] [Google Scholar]
  17. Petersheim M., Turner D. H. Base-stacking and base-pairing contributions to helix stability: thermodynamics of double-helix formation with CCGG, CCGGp, CCGGAp, ACCGGp, CCGGUp, and ACCGGUp. Biochemistry. 1983 Jan 18;22(2):256–263. doi: 10.1021/bi00271a004. [DOI] [PubMed] [Google Scholar]
  18. Puglisi J. D., Tinoco I., Jr Absorbance melting curves of RNA. Methods Enzymol. 1989;180:304–325. doi: 10.1016/0076-6879(89)80108-9. [DOI] [PubMed] [Google Scholar]
  19. Rastinejad F., Lu P. Bacteriophage T7 RNA polymerase. 19F-nuclear magnetic resonance observations at 5-fluorouracil-substituted promoter DNA and RNA transcript. J Mol Biol. 1993 Jul 5;232(1):105–122. doi: 10.1006/jmbi.1993.1373. [DOI] [PubMed] [Google Scholar]
  20. Sowers L. C., Eritja R., Kaplan B. E., Goodman M. F., Fazakerley G. V. Structural and dynamic properties of a fluorouracil-adenine base pair in DNA studied by proton NMR. J Biol Chem. 1987 Nov 15;262(32):15436–15442. [PubMed] [Google Scholar]
  21. Spiegelman S., Sawyer R., Nayak R., Ritzi E., Stolfi R., Martin D. Improving the anti-tumor activity of 5-fluorouracil by increasing its incorporation into RNA via metabolic modulation. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4966–4970. doi: 10.1073/pnas.77.8.4966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stolarski R., Egan W., James T. L. Solution structure of the EcoRI DNA octamer containing 5-fluorouracil via restrained molecular dynamics using distance and torsion angle constraints extracted from NMR spectral simulations. Biochemistry. 1992 Aug 11;31(31):7027–7042. doi: 10.1021/bi00146a003. [DOI] [PubMed] [Google Scholar]
  23. Takimoto C. H., Voeller D. B., Strong J. M., Anderson L., Chu E., Allegra C. J. Effects of 5-fluorouracil substitution on the RNA conformation and in vitro translation of thymidylate synthase messenger RNA. J Biol Chem. 1993 Oct 5;268(28):21438–21442. [PubMed] [Google Scholar]
  24. Weckbecker G. Biochemical pharmacology and analysis of fluoropyrimidines alone and in combination with modulators. Pharmacol Ther. 1991;50(3):367–424. doi: 10.1016/0163-7258(91)90051-m. [DOI] [PubMed] [Google Scholar]

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

RESOURCES