Degradation of nuclease-stabilized RNA oligonucleotides in Mycoplasma-contaminated cell culture media.

Journal Article (Journal Article)

Artificial RNA reagents such as small interfering RNAs (siRNAs) and aptamers often must be chemically modified for optimal effectiveness in environments that include ribonucleases. Mycoplasmas are common bacterial contaminants of mammalian cell cultures that are known to produce ribonucleases. Here we describe the rapid degradation of nuclease-stabilized RNA oligonucleotides in a human embryonic kidney 293 (HEK) cell culture contaminated with Mycoplasma fermentans, a common species of mycoplasma. RNA with 2'-fluoro- or 2'-O-methyl- modified pyrimidines was readily degraded in conditioned media from this culture, but was stable in conditioned media from uncontaminated HEK cells. RNA completely modified with 2'-O-methyls was not degraded in the mycoplasma-contaminated media. RNA zymogram analysis of conditioned culture media and material centrifuged from the media revealed several distinct protein bands (ranging from 30 to 68 kDa) capable of degrading RNA with 2'-fluoro- or 2'-O-methyl-modified pyrimidines. Finally, the mycoplasma-associated nuclease was detected in material centrifuged from the contaminated culture supernatants in as little as 15 minutes with an RNA oligo-containing 2'-O-methyl-modified pyrimidines and labeled with a 5'-fluorescein amidite (FAM) and 3'-quencher. These results suggest that mycoplasma contamination may be a critical confounding variable for cell culture experiments involving RNA-based reagents, with particular relevance for applications involving naked RNA (e.g., aptamer-siRNA chimeras).

Full Text

Duke Authors

Cited Authors

  • Hernandez, FJ; Stockdale, KR; Huang, L; Horswill, AR; Behlke, MA; McNamara, JO

Published Date

  • February 2012

Published In

Volume / Issue

  • 22 / 1

Start / End Page

  • 58 - 68

PubMed ID

  • 22229275

Pubmed Central ID

  • PMC3318257

Electronic International Standard Serial Number (EISSN)

  • 2159-3345

Digital Object Identifier (DOI)

  • 10.1089/nat.2011.0316


  • eng

Conference Location

  • United States