• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to previous page

In vitro selection of functional RNAs using 4'-thioNTP

Research Project

Project/Area Number 15510169
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Living organism molecular science
Research InstitutionHOKKAIDO UNIVERSITY

Principal Investigator

MINAKAWA Noriaki  Hokkaido Univ., Grad.School of Pharm.Sci., Asso.Prof., 大学院・薬学研究科, 助教授 (40209820)

Project Period (FY) 2003 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
Keywordsnucleoside / nucleotide / 4'-thioUTP / 4'-thioCTP / in vitro selection / RNA / aptamer / thrombin / 4'-thioRNA
Research Abstract

In vitro selection (or SELEX) is a biotechnological method used to isolate functional RNA molecules such as ribozymes and aptamers from a random pool of RNA combinatorial libraries. The aim of this work is to develop new NTP analogs for in vitro selection. The synthesis of the triphosphates of 4'-thioribonucleoside was achieved to give 4'-thioUTP, -CTP, -ATP, -GTP. Incorporation of 4'-thioUTP and 4'-thioCTP by T7 RNA polymerase to give 4'-thioRNA (thioRNA) proceeded well and was superior to those of the two sets of frequently used modified NTP analogs for in vitro selection (2'-NH_2dUTP and 2'-NH_2dCTP, and 2'-FdUTP and 2'-FdCTP), when an adequate leader sequence of DNA template was selected. I revealed that a leader sequence of about +15 of DNA template is important for effective incorporation of modified NTP analogs by T7 RNA polymerase. In addition, reverse transcription of the resulting thioRNA into the complementary DNA in the presence of 2'-deoxynucleoside triphosphates (dNTPs) also proceeded smoothly and precisely. The stability of the thioRNA toward RNase A was 50 times greater than that of the corresponding natural RNA. With these successful results in hand, I attempted the selection of thioRNA aptamers to human α-thrombin using 4'-thioUTP and 4'-thioCTP, and found a thioRNA aptamer with high binding affinity (K_d=4.7nM).

Report

(3 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report

Research Products

(19 results)

All 2005 2004 2003 Other

All Journal Article Publications

  • [Journal Article] 4'-Thionucleic Acids : Chemistry, Properties, and Applications for Developing Functional Oligonucleotides2005

    • Author(s)
      N.Minakawa et al.
    • Journal Title

      Frontiers in Organic Chemistry 1(In press)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] New NTP Analogs : The Synthesis of 4'-ThioUTP and 4'-ThioCTP and Their Utility for SELEX.2005

    • Author(s)
      Y.Kato et al.
    • Journal Title

      Nucleic Acids Res. (In press)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Synthesis and physical and physiological properties of 4'-thioRNA : application of post-modification of RNA aptamer toward NF-κB2004

    • Author(s)
      S.Hoshika et al.
    • Journal Title

      Nucleic Acids Res. 32

      Pages: 3815-3825

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] In vitro selection of human α-thrombin RNA aptamer using 4'-thioUTP and 4'-thioCTP2004

    • Author(s)
      Y.Kato et al.
    • Journal Title

      Nucleic Acids Res.Suppl. 48

      Pages: 223-224

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Synthesis and physical and physiological properties of 4'-thioRNA : application of post-modification of RNA aptamer toward NF-κB.2004

    • Author(s)
      S.Hoshika et al.
    • Journal Title

      Nucleic Acids Res. 32

      Pages: 3815-3815

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] In vitro selection of human α-thrombin RNA aptamer using 4'-thioUTP and 4'-thioCTP.2004

    • Author(s)
      Y.Kato et al.
    • Journal Title

      Nucleic Acids Res.Suppl. 48

      Pages: 223-223

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Synthesis and physical and physiological properties of 4'-thioRNA : application to post-modification of RNA aptamer toward NF-κB2004

    • Author(s)
      Hoshika, S. et al.
    • Journal Title

      Nucleic Acids Res. 32

      Pages: 3815-3825

    • Related Report
      2004 Annual Research Report
  • [Journal Article] In vitro selection of human α-thrombin RNA aptamer using 4'-thioUTP and 4'-thioCTP2004

    • Author(s)
      Kato, Y. et al.
    • Journal Title

      Nucleic Acids Res., Symp.Ser. 48

      Pages: 223-224

    • Related Report
      2004 Annual Research Report
  • [Journal Article] An Improved large scale synthesis of 1,4-anhydro-4-thio-D-ribitol.2003

    • Author(s)
      N.Minakawa et al.
    • Journal Title

      Tetrahedron 59

      Pages: 1699-1702

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Investigation of physical and physiological properties of 4'-thioribonucleotide (4'-thioRNA).2003

    • Author(s)
      S.Hoshika et al.
    • Journal Title

      Nucleic Acids Res.Suppl. 3

      Pages: 209-210

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] 4'-Thionucleic Acids : Chemistry, Properties, and Applications for Developing Functional Oligonucleotides.

    • Author(s)
      N.Minakawa et al.
    • Journal Title

      Frontiers in Organic Chemistry (in press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] New NTP Analogs : The Synthesis of 4'-ThioUTP and 4'-ThioCTP and Their Utility for SELEX.

    • Author(s)
      Y.Kato et al.
    • Journal Title

      Nucleic Acids Res. (in press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] 4'-Thionucleic Acids : Chemistry, Properties, and Applications for Developing Functional Oligonucleotides

    • Author(s)
      Minakawa, N. et al.
    • Journal Title

      Frontiers in Organic Chemistry (in press)

    • Related Report
      2004 Annual Research Report
  • [Publications] N.Minakawa et al.: "An Improved large scale synthesis of 1,4-anhydro-4-thio-D-ribitol"Tetrahedron. 59. 1699-1702 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] N.Minakawa et al.: "New Base Paring Motifs. The Synthesis and Thermal Stability of Oligodeoxynucleotides Containing Imidazopyridopyrimidine Nucleosides with the Ability to Form Four Hydrogen Bonds"J.Am.Chem.Soc.. 125. 9970-9982 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] N.Minakawa et al.: "A Versatile Modification of On-Column Oligodeoxynucleotides Using a Copper-Catalyzed Oxidative Acetylenic Coupling Reaction"J.Am.Chem.Soc.. 125. 11545-11525 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] A.B.Smith, III et al.: "Tremorgenic Indole Alkaloids. The Total Synthesis of (-)-Penitrem D"J.Am.Chem. Soc.. 125. 8228-8237 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] M.Tanimoto et al.: "No Enhancement of Nuclear Entry by Direct Conjugation of a Nuclear Localization signal Peptide to Linearized DNA"Bioconjugate Chem.. 14. 1197-1202 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] S.Hoshika et al.: "Investigation of physical and physiological properties of 4'-thioribonucleotide (4'-thioRNA)"Nucleic Acids Res.Suppl.. 3. 209-210 (2003)

    • Related Report
      2003 Annual Research Report

URL: 

Published: 2003-03-31   Modified: 2016-04-21  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi