A small world gets bigger

MicroRNAs (miRNAs) are ∼22-nucleotide non-coding RNA molecules that inhibit the translation or induce the degradation of protein-coding mRNAs in plants and animals. Recently, a member of the human herpesvirus family — Epstein–Barr virus — was found to encode miRNAs. These miRNAs do not have close homologues in other viral genomes or in the genome of their host, so it is difficult to identify other viral miRNAs using the available prediction software. Now, in Nature Methods, Tuschl and colleagues describe the development of a new computational method to identify miRNAs.

Their method predicts the probable location of miRNA precursors in a genome using information about local sequence composition and the predicted secondary structure of RNA transcripts (it assumes that miRNA precursors have specific sequence and structural features that are recognized by the processing enzymes). They applied this method to a subset of pathogenic DNA and RNA viruses (herpesviruses and small-genome RNA viruses, respectively) and, when possible, using their results as a guide, they cloned and sequenced small RNAs from virus-infected cells. As a result, they confirmed their prediction of many miRNAs in herpesviruses and, consistent with low prediction scores, they could not experimentally identify miRNAs in small-genome RNA viruses. This technique is therefore a good starting point for the identification of viral miRNAs. REFERENCEPfeffer, S. et al. Identification of microRNAs of the herpesvirus family. Nature Meth. 16 Feb 2005 (10.1038/nmeth746)

A restricted reaction

Single-molecule techniques have given us insights into molecular behaviours that often go unobserved in multiple-molecule experiments. With respect to enzymatic studies, single-molecule techniques can be used to monitor conformational changes in enzymes or binding/release events, but they are blind to the products of enzymatic reactions. One way to allow the products to be considered would be to inhibit their diffusion by carrying out the experiments in very small containers, and Noji and co-workers now describe suitable containers in Nature Biotechnology.

They used poly-dimethylsiloxane (PDMS), a silicone elastomer, to build a device that contains a large array of micrometre-sized cavities. By enclosing a liquid solution between this micropatterned sheet and a glass microscope slide, they could form femtolitre water volumes that were stable over long periods of time. PDMS is sufficiently impermeable to prevent evaporation or leakage for the duration of a typical bioassay, and the sheet ensures that the chambers have a uniform size. The authors proved that their device can be used to monitor the individual activities of isolated single enzymes — in particular, to monitor reaction products — by studying β-galactosidase and horseradish peroxidase as models. When trapped in a femtolitre chamber, a single molecule of each of these enzymes only needed a few turnovers to accumulate detectable concentrations of their products. This technique should therefore be useful for “...many ultrasensitive bioassays at the single-molecule level”. REFERENCERondelez, Y. et al. Microfabricated arrays of femtoliter chambers allow single molecule enzymology. Nature Biotechnol. 20 Feb 2005 (10.1038/nbt1072)