Enzymatic Production of Single-Molecule FISH and RNA Capture Probes

RNA. 2017 Oct;23(10):1582-1591. doi: 10.1261/rna.061184.117. Epub 2017 Jul 11.

Abstract

Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.

Keywords: RNA affinity purification; dideoxy-UTP; oligonucleotide labeling; single-molecule FISH; terminal deoxynucleotidyl transferase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biotin
  • DNA Nucleotidylexotransferase / metabolism*
  • Dideoxynucleotides / chemistry
  • Dideoxynucleotides / metabolism
  • Drosophila melanogaster / genetics
  • Female
  • Fluorescent Dyes / chemistry
  • In Situ Hybridization, Fluorescence / methods*
  • Oligonucleotide Probes / chemistry
  • Oligonucleotides / chemistry
  • Ovary / physiology
  • RNA Probes / chemistry*
  • RNA Probes / metabolism
  • Uracil Nucleotides / chemistry
  • Uracil Nucleotides / metabolism

Substances

  • Dideoxynucleotides
  • Fluorescent Dyes
  • Oligonucleotide Probes
  • Oligonucleotides
  • RNA Probes
  • Uracil Nucleotides
  • Biotin
  • 2',3'-dideoxyuridine-5'-triphosphate
  • DNA Nucleotidylexotransferase