To evaluate the stability of RNA with 2′-fluoro modified pyrimidines in conditioned culture media, a 51-nucleotide RNA with 2′-fluoro modified pyrimidines and a 3′-FAM was incubated in serum-containing media, conditioned with an HEK cell culture recently obtained from ATCC, or with an older HEK cell culture (). As previously reported, this RNA was found to be resistant to nuclease degradation in the presence of animal serum (unconditioned media incubation; lane 1 in ). While the modified RNA was also stable in conditioned media from the HEK cells obtained from ATCC, the conditioned media from the older HEK cells almost completely degraded it after a 4-hour incubation (lane 2, ). A PCR assay for the presence of mycoplasma detected a DNA sequence conserved within the genome of the mycoplasma genus in the culture supernatant of the older HEK cell culture supernatant, but not in the media from the more recently obtained culture (). A DNA sequence specific to the M. fermentans species was also detected in media from the older HEK cell culture, as indicated with a PCR product of expected size (lane 3, ). Sequencing of this PCR product confirmed that the amplified sequence is derived from the M. fermentans genome (not shown). In contrast, neither M. hominis nor M. penetrans was detected. As an additional means of detecting mycoplasma, DAPI staining of HEK cells from these 2 cultures was carried out (). Small, punctate, extra-nuclear DAPI labeling, indicative of mycoplasma contamination, was seen throughout the older HEK culture (), but only nuclear labeling was seen in the culture obtained from ATCC (). Together, these data demonstrate the presence of a ribonuclease that readily degrades RNA with 2′-fluoro modified pyrimidines in a mycoplasma-contaminated cell culture, but not in a mycoplasma-free HEK culture.
FIG. 1. An RNA oligo with 2′-fluoro-modified pyrimidines is degraded in conditioned media from mycoplasma contaminated HEK cells. A 51 nucleotide-long RNA oligo, composed of 2′-fluoro modified pyrimidines and unmodified purines was incubated with (more ...)
FIG. 2. Polymerase chain reaction (PCR)-based assay detected mycoplasma contamination in HEK cell culture and identified species as M. fermentans. PCR primer sets specific for genomic components of M. fermentans, Mycoplasma hominis, Mycoplasma penetrans, or for (more ...)
FIG. 3. Mycoplasma contamination detected in HEK cells with DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride) stain. HEK cells from 2 cultures—one older culture (A) and one recently obtained from ATCC (B)—were plated on glass-bottomed (more ...)
Next we studied the activity of this ribonuclease after heat pretreatment, in the presence of EDTA (a chelator of divalent cations), and in the presence of Superase.in (a broad-spectrum ribonuclease inhibitor) (). The same RNA oligo initially examined () was used for this experiment (51-mer with 2′-fluoro-modified pyrimidines and a 3′-FAM), with conditioned media from the mycoplasma-contaminated HEK cells. Results of this experiment indicate that the ribonuclease activity is sensitive to heat treatment (lane 3 in ) and is thus likely protein in nature. Like many ribonucleases, its activity is dependent on divalent cations, as chelation of divalent cations with EDTA inhibited degradation of the modified RNA (lane 4, ). However, the broad-spectrum RNase inhibitor, Superase.in, did not have an apparent impact on the activity (lane 5, ).
FIG. 4. Nuclease activity in mycoplasma-contaminated culture media is sensitive to heat treatment and depends on the presence of divalent cations. A 51-nucleotide-long RNA oligo, composed of 2′-fluoro modified pyrimidines and unmodified purines was incubated (more ...)
While the 2′-fluoro nucleotide modification is widely used for the development of RNA aptamer-based therapeutic approaches, other modifications are more commonly used to protect synthetic RNAs in other applications such as RNA interference (RNAi). The 2′-O-methyl modification is widely employed, and thus we examined the susceptibility of RNA with 2′-O-methyl-modified nucleotides to degradation by the mycoplasma-associated ribonuclease activity. For this experiment, 3 RNA oligos were used. Each was 51 nucleotides in length, of identical sequence, and with a 3′-FAM. The first oligo had 2′-fluoro-modified pyrimidines (purines were unmodified; described above), the second had 2′-O-methyl-modified pyrimidines (purines were unmodified), and every nucleotide of the third oligo was modified with 2′-O-methyls. Coincubation of these oligos with media conditioned by the mycoplasma-contaminated HEK cells for 30 minutes, 1 hour, 2 hours, or 4 hours again demonstrated the near-complete degradation of the oligo with 2′-fluoro-modified pyrimidines (lanes 1 and 2 in ). The oligo with 2′-O-methyl-modified pyrimidines was more resistant to degradation, but was almost completely degraded after 4 hours (lanes 3 and 4 in ). Finally, there was no detectable degradation of the oligo that was completely modified with 2′-O-methyls at any of the time-points (lanes 5 and 6, ).
FIG. 5. Time-course and substrate specificity of nuclease activity in mycoplasma contaminated culture media. Three 51-nucleotide RNA oligos of identical sequence but with different chemical modifications (2′-fluoro modified pyrimidines only, 2′-O-methyl (more ...)
Additional cell lines, contaminated with distinct mycoplasma species (i.e., non-M. fermentans species) were also tested for nuclease activity against RNA oligos with 2′-O-methyl- and 2′-fluoro- modified pyrimidines. Some of these cell lines possessed strong nuclease activity in their supernatants while others did not (not shown).
To further characterize the mycoplasma-associated ribonuclease, we carried out zymograms with unmodified DNA or RNA with 2′-fluoro- or 2′-O-methyl- modified pyrimidines (). For these experiments, we used serum-free conditioned cell culture media that was concentrated with filter centrifugation, as well as detergent-lysed particulate matter centrifuged from the conditioned media. The particulate matter presumably contains mycoplasma in the contaminated culture sample and was also found to possess ribonuclease activity.
FIG. 6. Nuclease sizes determined with zymograms. Concentrated media or material pelleted by centrifugation from media conditioned by mycoplasma-free or mycoplasma-contaminated HEK cells was resolved on 8% acrylamide-sodium dodecyl sulfate (SDS) gels embedded (more ...)
Multiple protein bands present in the mycoplasma-contaminated but not the mycoplasma-free concentrated culture supernatants could be seen in all 3 zymograms (lanes 2 and 3 in ). A cluster of bands that migrated between the 37- and 50-kDa molecular weight markers was prominent in these samples. A smaller protein, of approximately 30
kDa, digested the modified RNAs; no digestion was seen in this region of the DNA zymogram. A larger protein, of approximately 68
kDa, produced a band in the DNA zymogram, but not in the modified RNA zymograms. However, longer digestion periods (e.g., overnight) did yield a band of approximately this size in the modified RNA zymograms (not shown). The prominent cluster of bands between 37
kDa and 50
kDa present in all of the zymograms suggests the presence of multiple nucleases with broad substrate specificities.
The detergent-lysed particulate matter produced a very different pattern of bands on the zymograms (lanes 4 and 5, ). As with the concentrated supernatant, dark bands indicating digestion were only seen in the sample prepared from the mycoplasma-contaminated culture; however, the patterns of bands clearly indicate that there are multiple nucleases present in the particulate matter of the culture media with distinct substrate specificities. The prominent cluster of bands between the 37-kDa and 50-kDa molecular weight markers that was present in the concentrated culture supernatant was not seen in the particulate matter samples.
Because the mycoplasma-associated nuclease can be distinguished from endogenous mammalian nucleases by its distinct substrate specificity, we reasoned that the presence of mycoplasmas, in various contexts, might be inferred by the susceptibility of chemically modified ribonuclease substrates to degradation. While gel-based assays provide a simple and straightforward means of detecting nuclease activity, a more rapid and sensitive assay for ribonucleases that degrade unmodified RNA has been described. The basis for this assay is a short oligonucleotide RNase substrate, end-labeled with a fluorophore on one end that is rendered nonfluorescent by its close proximity to a quencher on the other end () (Kelemen et al., 1999
). Upon cleavage of the substrate, the quencher diffuses away from the fluorophore, which then exhibits fluorescence.
FIG. 7. Rapid detection of mycoplasma-associated nuclease activity with chemically modified RNase substrates. (A) The basis for nuclease detection with RNase substrates is illustrated. RNA oligonucleotides (5′-UCUCGUACGUUC-3′) with chemically (more ...)
This approach was adapted to detect the mycoplasma-associated nuclease by generating chemically modified RNase substrates with fluorophore and quencher conjugates. Four different RNA chemistries were tested: 2′-fluoro-modified pyrimidines, 2′-O-methyl-modified pyrimidines, complete 2′-fluoro modifications, and complete 2′-O-methyl modifications. Initially, each of these RNase substrates was incubated for 4 hours with culture media conditioned by either mycoplasma-free or mycoplasma-contaminated HEK cells (). While the complete 2′-O-methyl-modified substrate was not digested in either media, the other 3 RNase substrates exhibited substantially greater fluorescence after incubation in the mycoplasma-contaminated media. Of these, the substrate with 2′-O-methyl-modified pyrimidines exhibited the greatest relative fluorescence increase between uncontaminated and contaminated media. This substrate was thus characterized further.
Centrifugation of particulate matter from the mycoplasma-contaminated culture supernatants provided a simple and rapid means of obtaining concentrated nuclease activity. We explored the possibility that this approach might increase the sensitivity of mycoplasma detection with RNase substrates. The RNase substrate with 2′-O-methyl-modified pyrimidines was incubated with a detergent lysate of centrifuged particulate matter from mycoplasma-free or mycoplasma-contaminated HEK cells for 1 hour (). Conditioned media from these cultures was compared in parallel. While both the lysate and conditioned media exhibited strong nuclease activity, the lysate produced a greater signal in this assay. A clear signal could be seen over the background in this assay, even with an abbreviated (15 minutes) incubation (, fluorescence was measured on an ultraviolet light box in this case).
The contaminated HEK cell culture is a complex preparation, as it contains cells derived from two distinct organisms. While the uncontaminated HEK cells lack nuclease activity capable of efficiently degrading RNA with 2′-fluoro-modified or 2′-O-methyl-modified pyrimidines, we also sought to measure such activity in a pure culture of M. fermentans. Consistent with our observations of the contaminated HEK cell culture, lysates prepared from M. fermentans bacteria exhibited robust nuclease activity against RNA substrates with 2′-fluoro-modified and 2′-O-methyl-modified pyrimidines (). Nuclease activity against RNA substrates with 2′-fluoro-modified and 2′-O-methyl-modified pyrimidines was also found in the bacterial culture supernatant (not shown).
FIG. 8. Nuclease activity in a Mycoplasma fermentans lysate. RNase substrates with the indicated compositions were incubated with a lysate of M. fermentans bacteria for 1 hour at 37°C, and fluorescence levels were measured with a fluorescence plate reader. (more ...)