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Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
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Published on: August 9, 2019

RNA aptamer binding to polyhistidine-tag.

Shoutaro Tsuji1, Taku Tanaka, Naomi Hirabayashi

  • 1Division of Cancer Therapy, Kanagawa Cancer Center Research Institute, Yokohama-shi, Kanagawa 241-0815, Japan. stsuji@gancen.asahi.yokohama.jp

Biochemical and Biophysical Research Communications
|June 13, 2009
PubMed
Summary

Researchers developed RNA aptamers, named shot47, that bind specifically to the polyhistidine-tag (His-tag). This His-tag binder offers a cost-effective alternative for protein purification and analysis in biochemical applications.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Polyhistidine-tag (His-tag) is widely used for recombinant protein purification.
  • Existing methods like Ni2+-affinity purification are inhibited by divalent cations.
  • Antibody-based purification leads to antibody peptide contamination, hindering downstream analyses.

Purpose of the Study:

  • To isolate novel RNA aptamers that specifically bind to His-tag.
  • To evaluate the efficacy of these aptamers as alternatives to current His-tag purification and detection methods.

Main Methods:

  • Selection of RNA aptamers through SELEX (Systematic Evolution of Ligands by Exponential Enrichment).
  • Characterization of aptamer binding affinity using techniques like Surface Plasmon Resonance (SPR) or similar assays.
  • Application of the selected aptamer in various biochemical assays including ELISA, immunoprecipitation, and Western blotting.

Main Results:

  • Isolation of a high-affinity RNA aptamer, designated shot47, with a low picomolar dissociation constant for His-tag.
  • Demonstration that shot47 effectively binds His-tagged proteins even in the presence of divalent cations.
  • Successful application of shot47 as a substitute for antibodies in ELISA, immunoprecipitation, and Western blotting.

Conclusions:

  • The RNA aptamer shot47 is a potent and specific binder for His-tag.
  • Shot47 provides a robust alternative to antibody-based detection and purification, overcoming limitations of Ni2+-affinity methods.
  • Easy synthesis via in vitro transcription makes shot47 a cost-effective tool for biochemical analyses.