Functional Analysis of Noncoding RNAs in Trypanosomes: RNA Walk, a Novel Approach to Study RNARNA Interactions Between Small RNA and Its Target

The recent discovery of thousands of small noncoding RNAs (ncRNAs), in many different organisms, has led to the need for methods to study their function. One way to help understand their function is to determine what other RNAs interact with the ncRNAs. We have developed a novel method to investigate the RNA–RNA interactions between a small RNA and its target that we termed “RNA walk.” The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT-PCR. Domains carrying the cross-linked adducts are less efficiently amplified than domains that are not cross-linked. Real-time PCR is used to quantify the results. Further mapping of the interactions is performed by primer extension to determine the exact cross-linked adduct.