Nature Methods

A new method for the quick addition of small labeling molecules promises to simplify studies tracking the localization and dynamics of cell-surface proteins without interfering with cellular function.

Bigger isn't better when it comes to molecular tags. Visualization tools like green fluorescent protein (GFP) have proven invaluable in a wide variety of studies, but tacking on an entire protein adds bulk that can interfere with the very processes one is trying to observe. Smaller tags exist, but many of these also suffer from flaws such as rapid dissociation or excessive background.

Alice Ting and her colleagues have developed a different approach, taking advantage of a bacterial protein, BirA, which normally engineers the addition of a molecule called biotin to what is known as an acceptor peptide (AP) sequence. BirA can also be used to tack on biotin derivatives, and Ting's group developed a modified biotin with a unique chemical group not found in mammalian cells. This group becomes the platform for a specific chemical reaction that irreversibly attaches appropriately designed fluorescent tag molecules. Virtually any cell-surface protein containing the AP sequence can be labeled by this two-step process, which takes about twenty minutes.

Ting's group initially demonstrates that this labeling is highly specific and capable of detection that even outshines other fluorescence-based strategies; further tests with an AP-tagged cell-surface receptor protein reveal similar specificity and clarity, and show that tagging does not appear to interfere with the function of the receptor protein.

This technology offers an effective and less disruptive strategy for marking cell surface proteins, an approach Ting and her colleagues hope will soon be expanded for use in proteins inside the cell as well.

Nature Methods

New data from the rigorous analysis of a variety of viral genomes may provide important new insights into the mechanisms of viral pathogenesis, reports an article from Nature Methods to be published online on Wednesday 16 February.

Recent years have seen a sharp growth in awareness of the importance of microRNAs (miRNAs); small RNA molecules that, rather than coding for proteins, instead triggers the silencing of specific target genes. Numerous miRNAs have been identified in plants, invertebrates, and vertebrates. However, it was only recently - in the laboratory of Thomas Tuschl - that these molecules were also found in viruses, suggesting that this regulatory tactic could also be exploited to assist in the process of infection and replication.

Tuschl and his colleagues, including the group of Swiss researcher Mihaela Zavolan, announce their latest advance in this research front: a combined computational and experimental strategy for the identification of viral miRNAs. The first phase of this strategy involved identifying likely characteristics of viral miRNAs, and then developing an algorithm to apply these guidelines to locate appropriate genomic sequences. They analyzed a wide variety of human pathogenic viruses using this computational approach, and then experimentally confirmed their findings by cloning the small RNA molecules produced by cells infected with these viruses.

They identified and confirmed a number of novel miRNAs expressed by various DNA viruses, particularly among members of the herpesvirus family; at the same time, the RNA viruses that they investigated, including HIV-1 and hepatitis C virus, did not appear to produce miRNAs at all. Tuschl and Zavolan conclude that although a number of viral miRNAs may remain to be discovered, the new sequences identified by this strategy could lend powerful new insights into the process and prevention of viral pathogenesis.