Precisely biotinylated RNA

Experiments with RNA often require immobilization of RNA molecules, which is usually achieved by labeling with biotin for immobilization on avidin supports. Using current techniques, one can incorporate biotinylated ribonucleotide monophosphates (for example, UMP or CMP) into RNAs, or only tag the RNA 5' end by using a biotin derivative of the 5' ribonucleotide monophosphate (for example, GMP for the conventional T7 promoter) in a transcription reaction. One can also chemically modify the 5' or 3' end of purified RNA. The simplest approach, of course, is to incorporate the label during transcription, but for some experiments it is important to site-specifically label the RNA at a location other than the 5' end or to include only a single label to avoid altering the function of the RNA.

To achieve this, Ichiro Hirao and his colleagues at the University of Tokyo and at RIKEN modified unnatural base pairs such that a biotinylated base can be site-specifically incorporated into RNA by T7 RNA polymerase. For example, 2-amino-6-(2-thienyl)purine (s) can be incorporated into a DNA template. Then, in a standard transcription reaction, 2-oxo-(1H)pyridine (y) that had been biotinylated incorporates into the RNA transcript at sites complementary to s. This method can be easily adapted by any laboratory and can be used with commercially available transcription kits using T7 RNA polymerase. According to Hirao, "the available kits can be used without any changes of the protocol, except for using DNA templates containing unnatural bases such as s and substrates of y or modified y."

In work described in a recent Nucleic Acids Research article, the group applied this method to biotinylate and immobilize an anti−Raf-1 RNA aptamer on sensor chips; this aptamer accurately recognized its target, the Ras binding domain of Raf-1, which was fused to a glutathione S-transferase reporter.

Hirao believes this unnatural base pair system will be very useful for RNA-based technologies. If the unnatural base pairs work with both prokaryotic and eukaryotic RNA polymerases, the system could be applied in a wider range of studies, including in vivo experiments. Hirao also plans to extend this system to function in replication, transcription and translation: "If the DNA fragments containing the unnatural base pair can be amplified by PCR, the unnatural base pair system will be a much more powerful tool."