Probe Detects Single Copy Of Viral RNA

A new single-stranded DNA probe lights up when it binds just one copy of viral RNA inside a cell (Anal. Chem., DOI: 10.1021/ac3023873). Such a probe could help biologists study the progression of viral infections in cells and understand how drugs disrupt the infections, say the researchers who designed it.

The new method uses fluorescence in situ hybridization (FISH), a common technique for identifying nucleic acids inside cells or tissue. In FISH, scientists add chemicals to cells to freeze biomolecules in place. Then they apply to the cells a nucleic acid probe marked with a radioactive or fluorescent label. This nucleic acid contains a sequence complementary to nucleic acids that researchers are looking for. So after the researchers wash the cells, the only probe remaining in the cell is bound to the nucleic acids of interest. The researchers can detect its fluorescent or radioactive signals.

But the long DNA probes used in FISH often bind to other nucleic acids in the cell, limiting its detection ability.

Yong Chen, at the University of California, Los Angeles, and his colleagues wanted to design a more sensitive assay. They built a long single-stranded DNA containing about 1,000 copies each of two sequences: one complementary to a portion of the RNA that makes up the influenza A virus and another complementary to a fluorescently labeled strand of DNA. They designed the sequences so that they didn’t complement sequences found in their target cells.

To test their new probe, the scientists added it to dog kidney cells infected with influenza A. They had chemically frozen the cells’ biomolecules before adding the probe. After washing away unbound probe, they added the fluorescently labeled strand of DNA.

When the researchers looked at the cells under a microscope, single molecules of viral RNA appeared as bright red dots. They assumed their probes bound a single RNA molecule because the viral nucleic acid concentrations early in the infection were much smaller than that of their DNA probe. The scientists didn’t see the glowing spots in uninfected cells, indicating very little off-target binding, Chen says. His team repeated the experiment with infected cells treated with the antiviral drug ribavirin. Six hours after infection, the amount of viral RNA in those cells was one-twelfth that of untreated cells.