Structure analysis of microRNA precursors.

MicroRNA biogenesis occurs in several steps from their precursors having irregular hairpin structures. The highly variable architecture of these stem-and-loop structures, which have terminal loops of various sizes and diverse structure destabilizing motifs present in their stem sections, may strongly influence the process of microRNA liberation. In order to better understand this process, more details regarding its structural basis are required. A substantial part of this information may be derived from the structure analysis of microRNA precursor using biochemical methods. Here we show how the analysis with the use of various nucleases and metal ions is performed. The presented protocols include the design of DNA template-phage promoter fusions to generate natural precursor ends, and the tests performed to check the sequence and structure homogeneity of the in vitro transcripts prior to probing their structures.