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Reactivity-dependent PCR: direct, solution-phase in vitro selection for bond formation.

David J Gorin1, Adam S Kamlet, David R Liu

  • 1Howard Hughes Medical Institute and Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

Journal of the American Chemical Society
|June 16, 2009
PubMed
Summary

Researchers developed reactivity-dependent PCR (RDPCR) to directly select functional nucleic acids and DNA-encoded molecules. This new method simplifies selections for bond-forming and bond-cleaving reactions, eliminating complex steps.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • In vitro selection is crucial for discovering functional nucleic acids and DNA-encoded small molecules.
  • Current methods for selecting bond-forming/cleaving reactivity are indirect and labor-intensive, involving multiple steps.

Purpose of the Study:

  • To develop and validate a novel method, reactivity-dependent PCR (RDPCR), for direct selection of nucleic acid and DNA-encoded molecule reactivity.
  • To streamline the process of identifying molecules with specific catalytic or cleavage activities.

Main Methods:

  • Development of reactivity-dependent PCR (RDPCR), a method linking bond formation/cleavage to sequence amplification.
  • Elimination of solid-phase capture, washing, and elution steps required in traditional selection methods.
  • Application of RDPCR for both reaction discovery (bond formation) and protease activity profiling (bond cleavage).

Main Results:

  • Successful development and validation of RDPCR for direct selection.
  • Demonstration of RDPCR's utility in identifying bond-forming reactions.
  • Validation of RDPCR for protease activity profiling and selection of bond-cleaving molecules.

Conclusions:

  • RDPCR offers a more direct and efficient approach for in vitro selection of functional nucleic acids and DNA-encoded molecules.
  • The method simplifies the discovery of novel reactions and the profiling of enzymatic activities.
  • RDPCR obviates the need for complex multi-step manipulations, accelerating the discovery process.