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Modified nucleic acids for in vitro selection

Y Ito1

  • 1Graduate School of Material Science, Kyoto, Japan.

Nucleic Acids Symposium Series
|January 1, 1997
PubMed
Summary
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Modified nucleotides can be used in in vitro evolution techniques like SELEX. Researchers found specific modified nucleoside triphosphates are accepted as substrates for transcription, enabling in vitro selection applications.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • In vitro evolution methods, such as Systematic Evolution of Ligands by Exponential Enrichment (SELEX), are crucial for discovering novel nucleic acid aptamers.
  • The efficiency of SELEX relies on the ability of polymerases to transcribe modified nucleotide triphosphates incorporated into DNA templates.

Purpose of the Study:

  • To investigate the compatibility of various modified nucleoside triphosphates with T7 RNA polymerase for in vitro transcription.
  • To determine which modified nucleotides can be successfully incorporated and transcribed, thereby expanding the toolkit for in vitro selection.

Main Methods:

  • Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was employed.
  • T7 RNA polymerase was used to transcribe DNA templates containing modified nucleoside triphosphates.

Related Experiment Videos

  • Reverse transcription was performed on the transcribed RNA products to generate cDNA templates.

    • Main Results:

    • T7 RNA polymerase accepted cytidine triphosphate with a biotinyl group (N4-position), adenosine triphosphate with an aminohexyl group (N6-position), and 2'-amino-2'-deoxyuridine triphosphate as substrates.
    • Uridine triphosphate with a nitroveratryloxy group (2'-position) and adenosine triphosphate with an aminohexyl group (8-position) were not accepted as substrates.
    • Successful transcription of modified nucleotides indicates their utility in subsequent SELEX rounds.

    Conclusions:

    • Specific modified nucleoside triphosphates are suitable substrates for T7 RNA polymerase-mediated transcription.
    • These findings demonstrate the potential of incorporating selected modified nucleotides into in vitro selection strategies.
    • The study expands the scope of modified nucleic acid applications in molecular evolution and aptamer discovery.