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Letters |
1 Department of Pathology and Laboratory Medicine Christiana Care Health System Newark, DE 19718
aAddress for correspondence: 236 Laurel Heights Rd., Landenberg, PA 19350. Fax 610-274-8896; e-mail ksisco2{at}ix.netcom.com.
To the Editor:
DNA and RNA are present in normal and diseased human plasma (1)(2)(3)(4)(5)(6)(7) and in the extracellular media of eukaryotic cell cultures (8)(9). Two excellent recent reviews (10)(11) indicated that neither necrotic nor apoptotic cells may fully explain the presence of DNA in plasma. In favor of apoptosis is the finding of a DNA ladder pattern after electrophoresis. Electrophoresis of nick-translated DNA isolated from plasma of healthy controls produced autoradiographic bands at sizes equivalent to whole-number multiples (1–5x) of nucleosomal DNA (185–200 bp) (12). Nevertheless, DNA is released in a homeostatic fashion (8)(9), and newly synthesized DNA is preferentially released (9)(13)(14), suggesting that neither apoptosis nor necrosis is the sole source of this DNA.
Cells not only release DNA but have specialized receptors for these nucleoprotein complexes as well. A recent study (15) showed that nucleosomes bind to the exterior of cultured mesangial cells in a receptor-mediated fashion. Competition studies demonstrated inhibition of binding by nucleosomes, 200/400-bp DNA, salmon sperm DNA, and, to a lesser extent, single-stranded DNA. DNase treatment removed 95.3% ± 2.3% of the cell-associated radioactivity at 37 °C. The authors view this as "essentially all cell-associated radioactivity", although a small percentage remained (2.4–7.0%) along with some remaining nuclear labeling by autoradiography, suggesting the possibility of uptake of labeled oligonucleosomes.
RNA has also been detected in plasma (2)(4)(5)(7)(16)(17), with the amount ranging from 25.4 to 144 mg/L (7)(17). It is resistant to RNases A and T1 and endogenous RNases (16). Some of the RNA is reportedly linked by hydrogen bonds to DNA, and its release from cultured cells is regulated by the same homeostatic mechanism that governs DNA release (9). The RNA in plasma consists predominantly of uridine bases with a small amount of guanidine. No adenine or cytosine was found after alkaline hydrolysis, followed by protein and lipid extraction and anion-exchange column chromatography (17). The authors of the article did not quantify the ratio of GMP and UMP, but using the area under the published peaks for UMP and GMP and applying the relevant extinction coefficients for 0.01 mol/L HCl solutions of these ribonucleotides, I estimate the UMP:GMP ratio as 
8:1. This suggests a special, possibly repetitive sequence in the RNA that may relate to its function.
I aimed to test in normal fasting human serum the findings of Stroun et al. (9), who found features of DNA-RNA heteroduplexes in oligonucleotides from their extracellular culture media. I exposed ethidium bromide-containing serum to RNase H, which selectively degrades RNA in RNA-DNA heteroduplex molecules. The fluorescence intensity of ethidium bromide changes with changes in the oligonucleotide base pairing (as in nuclease digestion) (18). Fluorescence measurements were made on a Perkin-Elmer 204-A Fluorescence Spectrophotometer at 25 °C, using 60 µmol/L (25 mg/L) ethidium bromide in normal human serum. The excitation and emission wavelengths were 564 and 590 nm, respectively.
Escherichia coli Ribonuclease H (free of detectable nonspecific DNase or RNase; Gibco BRL) was added at 3.3, 20, or 120 kU/L. I treated ethidium bromide-containing normal human serum with these three concentrations of RNase H. This endoribonuclease specifically degrades the RNA strand of a RNA-DNA hybrid to produce 5' phosphate-terminated oligoribonucleotides and single-stranded DNA. Serum was chosen instead of plasma because heparin and divalent cation binding agents are potent inhibitors of RNase H (19). Because ethidium bromide fluorescence is dependent on base pairing for the highest quantum yield, RNase H activity would be manifested by a decrease in fluorescence. With 500 mL/L glycerol in water as a control, dilutional effects on fluorescence were measured and were found to be equal to that predicted mathematically by simple volume dilution. A glycerol-water solution was used because the enzyme is dissolved in a storage buffer containing, per liter, 500 mL of glycerol, 20 mmol of Tris-HCl (pH 7.5), 100 mmol of KCl, 10 mmol of MgCl2, 0.1 mmol of EDTA, 0.1 mmol of dithiothreitol, and 50 mg of bovine serum albumin. Baseline fluorescence was measured for 10 min before the addition of enzyme, and the variation in fluorescence was <0.3%. The addition of RNase H produced a decrease in fluorescence after correction for dilutional effects as described above. This decrease was enzyme concentration dependent as shown in Table 1
. The decrease in fluorescence was complete in 5 min, and fluorescence varied <0.3% thereafter for 25 min, when the experiments were stopped. I conclude that at least some of the RNA present in the serum is in the form of a RNA-DNA heteroduplex.
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The data on DNA and RNA in plasma, serum, and extracellular culture media are consistent with the conclusion that oligonucleosomes are present in human plasma and serum. Part of the DNA of these nucleosomes may be in the form of a RNA-DNA heteroduplex with the RNA consisting of a predominantly polyuridine sequence (17). This structure becomes especially important if indeed these nucleosomes are taken up to a small extent by cells (15). Stroun et al. (9) found that "extracellular RNA" from lymphocytes stimulated in vitro DNA synthesis of monkey liver DNA with partially purified E. coli DNA polymerase at a rate up to 10-fold greater than that of controls without RNA. Whether a predominantly polyuridine RNA in the form of a RNA-DNA heteroduplex presumably in this system acts as a primer requires further analysis. Clearly, however, further work characterizing the DNA and RNA sequences of the serum nucleosomes would provide valuable information. If cellular uptake and expression could be demonstrated for plasma nucleosomes, these structures could serve as endogenous vehicles for genetic therapy.
References
The following articles in journals at HighWire Press have cited this article:
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  E. Schutz, H. B. Urnovitz, L. Iakoubov, W. Schulz-Schaeffer, W. Wemheuer, and B. Brenig Bov-tA Short Interspersed Nucleotide Element Sequences in Circulating Nucleic Acids from Sera of Cattle with Bovine Spongiform Encephalopathy (BSE) and Sera of Cattle Exposed to BSE Clin. Vaccine Immunol., July 1, 2005; 12(7): 814 - 820. [Abstract] [Full Text] [PDF]
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 T. El-Hefnawy, S. Raja, L. Kelly, W. L. Bigbee, J. M. Kirkwood, J. D. Luketich, and T. E. Godfrey Characterization of Amplifiable, Circulating RNA in Plasma and Its Potential as a Tool for Cancer Diagnostics Clin. Chem., March 1, 2004; 50(3): 564 - 573. [Abstract] [Full Text] [PDF]
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N. B.Y. Tsui, E. K.O. Ng, and Y.M. D. Lo Stability of Endogenous and Added RNA in Blood Specimens, Serum, and Plasma Clin. Chem., October 1, 2002; 48(10): 1647 - 1653. [Abstract] [Full Text] [PDF]
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