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DNA display of folded RNA libraries enabling RNA-SELEX without reverse transcription.

I S MacPherson1, J S Temme2, I J Krauss2

  • 1Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawai'i at Manoa, 651 Ilalo St., Biosciences Building, Suite 325, Honolulu, Hawaii 96813-5525, USA. iain.macpherson@hawaii.edu and Department of Chemistry, Brandeis University, 415 South St. MS 015, Waltham, MA 02454-9110, USA. kraussi@brandeis.edu.

Chemical Communications (Cambridge, England)
|February 22, 2017
PubMed
Summary

This study presents a novel method for directly linking RNA to its DNA template, bypassing the need for reverse transcription in RNA selection. This technique facilitates the discovery of modified RNA aptamers and ribozymes.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Synthetic Biology

Background:

  • Systematic evolution of RNA ligands by exponential enrichment (SELEX) typically requires a reverse transcription step.
  • This step can be challenging for RNA molecules with modified bases that resist accurate cDNA synthesis.
  • Developing alternative SELEX methods is crucial for expanding the scope of RNA-based therapeutics and diagnostics.

Purpose of the Study:

  • To present a novel method for physically attaching folded RNA libraries to their encoding DNA.
  • To circumvent the reverse transcription step in RNA-SELEX.
  • To enable the selection of RNA aptamers and ribozymes with modified bases.

Main Methods:

  • A DNA library is modified with an isodC base to stall T7 polymerase.
  • A 5' "capture strand" is incorporated, which anneals to the nascent RNA transcript.
  • This physically links the RNA product to its DNA template.

Main Results:

  • The method was successfully validated by selecting RNA aptamers against human α-thrombin.
  • The selected aptamers exhibited dissociation constants in the low nanomolar range.
  • The physical attachment strategy effectively bypassed the reverse transcription step.

Conclusions:

  • This physical attachment method offers a robust alternative to traditional RNA-SELEX.
  • It is particularly useful for discovering RNA aptamers and ribozymes containing modified bases.
  • This approach broadens the potential for developing novel RNA-based molecules with enhanced properties.