Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

Potent 2′-amino-, and 2′-fluoro-2′- deoxyribonucleotide RNA inhibitors of keratinocyte growth factor

Nature Biotechnology volume 15, pages 68–73 (1997)Cite this article

Abstract

Reiterative in vitro selection-amplification from random oligonucleotide libraries allows the identification of molecules with specific functions such as binding to specific proteins. The therapeutic usefulness of such molecules depends on their high affinity and nuclease resistance. Libraries of RNA molecules containing 2′amino-(2′NH2)- or 2′fluoro-(2′F)-2′-deoxypyrimidines could yield ligands with similar nuclease resistance but not necessarily with similar affinities. This is because the intramolecular helices containing 2′NH2 have lower melting temperatures (Tm) compared with helices containing 2′F, giving them thermodynamically less stable structures and possibly weaker affinities. We tested these ideas by isolating high-affinity ligands to human keratinocyte growth factor from libraries containing modified RNA molecules with either 2′NH2 or 2′F pyrimidines. We demonstrated that 2′F RNA ligands have affinities (Kd approximately 0.3–3 pM) and bioactivities (Ki approximately 34 pM) superior to 2′NH2 ligands (Kd approximately 400 pM and Ki approximately 10 nM). In addition, 2′F ligands have extreme thermostabilities (Tm approximately 78°C in low salt, and specificities).

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Gold, L. 1995. Oligonucleotides as research, diagnostic, and therapeutic agents. J. Biol. Chem. 270: 13581–13584.

    Article  CAS  Google Scholar 

  2. Gold, L., Polisky, B., Uhlenbeck, O., and Yarus, M. 1995. Diversity of oligonucleotide functions. Annu. Rev. Biochem. 64: 763–797.

    Article  CAS  Google Scholar 

  3. Green, L., Waugh, S., Binkley, J.P., Hostomska, Z., Hostomsky, Z., and Tuerk, C. 1995. Comprehensive chemical modification interference and nucleotide substitution analysis of an RNA pseudoknot inhibitor to HIV-1 reverse transcriptase. J. Mol. Biol. 247: 60–68.

    Article  CAS  Google Scholar 

  4. Klußmann, S., Nolte, A., Bald, R., Erdmann, V.E., and Furste, J.P. 1996. Mirror-image RNA that binds D-adenosine. Nature Biotechnology 14: 1112–1115.

    Article  Google Scholar 

  5. Nolte, A., Klußmann, S., Bald, R., Erdmann, V.E., and Furste, J.P. 1996. Mirror-design of L-oligonucleotide ligands binding to L-arginine. Nature Biotechnology 14: 1116–1119.

    Article  CAS  Google Scholar 

  6. Pieken, W.A., Olsen, D.B., Benseler, F., Aurup, H., and Eckstein, F. 1991. Kinetic characterization of ribonuclease-resistant 2′-modified hammerhead ribozymes. Science 253: 314–317.

    Article  CAS  Google Scholar 

  7. Pan, W., Craven, R.C., Qiu, Q., Wilson, C.B., Wills, J.W., Golovine, S. et al. 1995. Isolation of virus-neutralizing RNAs from a large pool of random sequences. Proc. Natl. Acad. Sci. USA 92: 11509–11513.

    Article  CAS  Google Scholar 

  8. Jellinek, D., Green, L.S., Bell, C., Lynott, C.K., Gill, N., Vargeese, C. et al. 1995. Potent 2′-amino-2′-deoxypyrimidine RNA inhibitors of basic fibroblast growth factor. Biochemistry 34: 11363–11372.

    Article  CAS  Google Scholar 

  9. Lin, Y., Qui, Q., Gill, S.C., and Jayasena, S.D. 1994. Modified RNA sequence pools for in vitro selection. Nucleic Acids Res. 22: 5229–5234

    Article  CAS  Google Scholar 

  10. Eaton, B.E., Gold, L., and Zichi, D.A. 1995. Let's get specific: the relationship between specificity and affinity. Chemistry & Biology 2: 633–638.

    Article  CAS  Google Scholar 

  11. Aurup, H., Tuschl, T., Benseler, F., Ludwig, J., and Eckstein, F. 1994. Oligonucleotide duplexes containing 2′-amino-2′-deoxycytidines: thermal stability and chemical reactivity. Nucleic Acids Res. 22: 20–24.

    Article  CAS  Google Scholar 

  12. Kawasaki, A.M., Casper, M.D., Freier, S.M., Lesnik, E.A., Zounes, M.C., Cummins, L.L. et al. 1993. Uniformly modified 2′-deoxy-2′fluoro phosphorothioate oligo-nucleotides as nuclease-resistant antisense compounds with high affinity and specificity for RNA targets. J. Med. Chem. 36: 831–841.

    Article  CAS  Google Scholar 

  13. Lesnik, E.A., Guinosso, C.J., Kawasaki, A.M., Sasmor, H., Zounes, M., Cummins, L.L. et al. 1993. Oligodeoxynecleotides containing 2′-O-modified adenosine: synthesis and effect on stability of DNA:RNA duplexes. Biochemistry 32: 7832–7838.

    Article  CAS  Google Scholar 

  14. Monia, B.P., Lesnik, E.A., Gonzalez, C., Lima, W.F., McGee, D., Guinosso, C.J. et al. 1993. Evaluation of 2′-modified Oligonucleotides containing 2′-deoxy gaps as antisense inhibitors of gene expression. J. Biol. Chem. 268: 14514–14522.

    CAS  PubMed  Google Scholar 

  15. Rubin, J.S., Osada, H., Finch, P.W., Taylor, W.G., Rudikoff, S., and Aaronson, S.A. 1989. Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc. Natl. Acad. Sci. USA 86: 802–806.

    Article  CAS  Google Scholar 

  16. Finch, P.W., Rubin, J.S., Miki, T., Ron, D., and Aaronson, S.A. 1989. Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science 244: 752–755.

    Article  Google Scholar 

  17. Miki, T., Bottaro, D.P., Fleming, T.P., Smith, C.L., Burgess, W.H., Chan, A.M.L., et al. 1992. Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene. Proc. Natl. Acad. Sci. USA 89: 246–250.

    Article  CAS  Google Scholar 

  18. Staiano-Coico, L., Krueger, J.G., Rubin, J.S., D'limi, S., Vallat, V.P., Valentine, L. et al. 1993. Human keratinocyte growth factor effect in a porcine model of epidermal wound healing. J. Ex. Med. 178: 865–878.

    Article  CAS  Google Scholar 

  19. Brauchle, M., Madlener, M., Wagner, A.D., Angermeyer, K., Lauer, U., Hofschneder, P.H. et al. 1996. Keratinocyte growth factor is highly overex-pressed in inflammatory bowel disease. Am. J. Pathol. 149: 521–529.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Finch, P.W., Pricolo, V., Wu, A., and Finkelstein, S.D. 1996. Increased expression of keratinocyte growth factor messenger RNA associated with inflammatory bowel disease. Gastroenterology 110: 441–451.

    Article  CAS  Google Scholar 

  21. Ishii, H., Hattori, Y., Itoh, H., Kishi, T., Yoshida, T., Sakamoto, H., et al. 1994. Preferential expression of the third immnoglobulin-like domain of K-sam product provides keratinocyte growth factor-dependent growth in carcinoma cell lines. Cancer Res. 54: 518–522.

    CAS  PubMed  Google Scholar 

  22. Katoh, M., Hattori, Y., Sasaki, H., Tanaka, M., Sugano, K., Yazaki, Y. et al. 1992. K-sam gene encodes secreted as well as transmembrane receptor tyrosine kinase. Proc. Natl. Acad. Sci. USA 89: 2960–2964.

    Article  CAS  Google Scholar 

  23. Yi, E.S., Yin, S., Harclerode, D.L., Bedoya, A., Bikhazi, N.B., Housley, R.M. et al. 1994. Keratinocyte growth factor induces pancreatic ductal epithelial proliferation. Am. J. Pathol. 145: 80–85.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Housley, R.M., Morris, C.F., Boyle, W., Ring, B., Biltz, R., Tarpley, J.E. et al. 1994. Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract. J. Clin. Invest. 94: 1764–1777.

    Article  CAS  Google Scholar 

  25. Ulich, T.R., Yi, E.S., Longmuir, K., Yin, S., Biltz, R., Morris, C.F. et al. 1994. Keratinocyte growth factor is a growth factor for type II pneumonocytes in vivo. J. Clin. Invest. 93: 1298–1306.

    Article  CAS  Google Scholar 

  26. Yi, E.S., Bedoya, A.A., Lee, H., Kim, S., Housley, R.M., Aukerman, S.L. et al. 1994. Keratinocyte growth factor causes cystic dilation of the mammary glands of mice. Interaction of keratinocyte growth factor, estrogen, and progesterone in vivo. Am. J. Pathol. 145: 1015–1022.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Ulich, T.R., Yi, E.S., Cardiff, R., Yin, S., Bikhazi, N., Biltz, R. et al. 1994. Keratinocyte growth factor is a growth factor for mammary epithelium in vivo. The mammary epithelium of lactating rats is resistant to the proliferative action of keratinocyte growth factor. Am. J. Pathol. 144: 862–868.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Yi, E.S., Shabaik, A.S., Lacey, D.L., Bedoya, A.A., Yin, S., Housley, R.M. et al. 1995. Keratinocyte growth factor causes proliferation of urothelium in vivo. J. Urol. 154: 1566–1570.

    Article  CAS  Google Scholar 

  29. Yan, G., Fukabori, Y., Nikolaropoulos, S., Wang, F., and McKeehan, W.L. 1992. Heparine-binding keratinocyte growth factor is a candidate stromal to epithelial cell andromedin. Mol. Endo. 6: 2123–2128.

    CAS  Google Scholar 

  30. Culig, Z., Hobisch, A., Cronauer, M.V., Radmayr, C., Trapman, J., Hittmair, A. et al. 1994. Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-l, keratinopyte growth factor, and epidermal growth factor. Cancer Res. 54: 5474–5478.

    CAS  PubMed  Google Scholar 

  31. Bottaro, D.P., Fortney, E., Rubin, J.S., and Aaronson, S.A. 1993. A keratinocyte growth factor receptor-derived peptide antagonist identifies part of the ligand binding site. J. Biol. Chem. 268: 9180–9183.

    CAS  PubMed  Google Scholar 

  32. Woese, C.R., and Pace, N.R. 1993. Probing RNA structure, function, and his-tory by comparative analysis, pp. 91–117 in The RNA world. Gesteland, R.F. and Atkins, J.F. (eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  33. Wyatt, J.R., and Tinoco, I. Jr. 1993. RNA structural elements and RNA function in pp. 465–496 The RNA world. Gesteland, R.F. and Atkins, J.F. (eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  34. ten Dam, E., 1992. Structural and functional aspects of RNA pseudoknots. Biochemistry 31: 1665–11676.

    Article  Google Scholar 

  35. Du, Z., Giedroc, D.P., and Hoffman, D.W. 1996. Structure of the autoregulatory pseudoknot within the gene 32 messenger RNA of bacteriophages T2 and T6: a model for a possible family of structurally related RNA pseudoknots. Biochemistry 35: 4187–4198.

    Article  CAS  Google Scholar 

  36. Qiu, H., Kaluarachchi, K., Du, Z., Hoffman, D.W., and Giedroc, D.P. 1996. Thermodynamics of folding of the RNA pseudoknot of the T4 gene 32 autoregulatory messenger RNA. Biochemistry 35: 4176–4186.

    Article  CAS  Google Scholar 

  37. Shen, L.X. and Tinoco, I. Jr., 1995. The structure of an RNA pseudoknot that causes efficient frameshifting in mouse mammary tumor virus. J. Mol. Biol. 247: 963–978.

    Article  CAS  Google Scholar 

  38. Tuerk, C., MacDougal, S., and Gold, L. 1992. RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase. Proc. Natl. Acad. Sci. USA 89: 6988–6992.

    Article  CAS  Google Scholar 

  39. Ringquist, S., Jones, T., Snyder, E.E., Gibson, T., Boni, I., and Gold, L. 1995. High-affinity RNA ligands to Escherichia coli ribosomes and ribosomal protein S1: comparison of natural and unnatural binding sites. Biochemistry 34: 3640–3648.

    Article  CAS  Google Scholar 

  40. Binkley, J., Alien, P., Brown, D.M., Green, L., Tuerk, C., and Gold, L., 1995. RNA ligands to human nerve growth factor. Nucleic Acids Res. 23: 3198–3205.

    Article  CAS  Google Scholar 

  41. Lorsch, J.R., and Szostak, J.W. 1994. In vitro selection of RNA aptamers specific for cyanocobalamin. Biochemistry 33: 973–982.

    Article  CAS  Google Scholar 

  42. Hilbers, C.W., Robillard, G.T., Shulman, R.G., Blake, R.D., Webb, P.K., Fresco, R. et al. 1976. Thermal unfolding of yeast glycine transfer RNA. Biochemistry 15: 1874–1882.

    Article  CAS  Google Scholar 

  43. Kaighn, M.E., Narayan, K.S., Ohnuki, Y., Lechner, J.F., and Jones, L.W. 1979. Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Investigative Urology 17: 166–23.

    Google Scholar 

  44. Weissman, B.E., and Aaronson, S.A. 1983. BALB and Kirsten murine sarcoma viruses alter growth and differentiation of EGF-dependent balb/c mouse epidermal keratinocyte lines. Cell 32: 599–606.

    Article  CAS  Google Scholar 

  45. Caputo, J.L., Hay, R.J., and Williams, C.D. 1979. The isolation and properties of an epithelial cell strain from rhesus monkey bronchus. In Vitro 15: 222–223.

    Google Scholar 

  46. Tuerk, C., and Gold, L. 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249: 505–510.

    Article  CAS  Google Scholar 

  47. Fitzwater, T., and Polisky, B. 1996. A SELEX primer. Methods Enzymol. 267: 275–301.

    Article  CAS  Google Scholar 

  48. Feng, D. and Doolittle, R.F. 1987. Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J. Mol. Evol. 25: 351–360.

    Article  CAS  Google Scholar 

  49. Jaeger, J.A., Turner, D.H., and Zuker, M. 1989. Improved predictions of secondary structures for RNA. Proc. Natl. Acad. Sci. USA 86: 7706–7710.

    Article  CAS  Google Scholar 

  50. Jaeger, J.A., Turner, D.H., and Zuker, M. 1990. Predicting optimal and suboptimal secondary structure for RNA. Methods Enzymol. 183: 281–306.

    Article  CAS  Google Scholar 

  51. Zuker, M. 1989. On finding all suboptimal folding of an RNA molecule. Science 244: 48–52.

    Article  CAS  Google Scholar 

  52. Jellinek, D., Lynott, C.K., Rifkin, D.B., and Janjic, N. 1993. High-affinity RNA lig-ands to basic fibroblast growth factor inhibit receptor binding. Proc. Natl. Acad. of Sci. USA 90: 11227–11231.

    Article  CAS  Google Scholar 

  53. Carey, J., Cameron, V., de-Haseth, P.L., and Uhlenbeck, O.C. 1983. Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site. Biochemistry 22: 2601–2610.

    Article  CAS  Google Scholar 

  54. Miki, T., Fleming, T.P., Bottaro, D.P., Rubin, J.S., Ron, D., and Aaronson, S.A. 1991. Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. Science 251: 72–75.

    Article  CAS  Google Scholar 

  55. Bottaro, D.P., Rubin, J.S., Ron, D., Finch, P.W., Florio, C., and Aaronson, S.A. 1990. Characterization of the receptor for keratinocyte growth factor. J. Biol. Chem. 265: 12767–12770.

    CAS  PubMed  Google Scholar 

  56. Gill, S.C., Weitzel, S.E., and von-Hippel, P.H., 1991. Escherichia coli σ70 and NusA proteins I. Binding interactions with core RNA polymerase in solution and within the transcription complex. J. Mol. Biol. 220: 307–324.

    Article  CAS  Google Scholar 

  57. Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

Download references

Author information

Author notes

  1. Derek Jellinek

    Present address: Children's Medical Research Institute, Locked Bag 23, Wentworthville, 2145, NSW, Australia

Authors and Affiliations

  1. NeXstar Pharmaceuticals, Inc., 2860 Wilderness PL, Boulder, CO, 80301

    Nikos C. Pagratis, Carol Bell, Ying-Fon Chang, Susan Jennings, Tim Fitzwater & Chinh Dang

Authors
  1. Nikos C. Pagratis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carol Bell
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ying-Fon Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Susan Jennings
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tim Fitzwater
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Derek Jellinek
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chinh Dang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nikos C. Pagratis.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pagratis, N., Bell, C., Chang, YF. et al. Potent 2′-amino-, and 2′-fluoro-2′- deoxyribonucleotide RNA inhibitors of keratinocyte growth factor. Nat Biotechnol 15, 68–73 (1997). https://doi.org/10.1038/nbt0197-68

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0197-68

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing