Analysis of RNA secondary structure.

Abstract

RNA has different levels of structural organization. The primary structure is the linear order of the nucleotide monomers, the RNA sequence. During transcription process, the partially synthesized RNA is folded by base-pairing and thermodynamic intramolecular or intermolecular interactions. This results in a dynamic spreading of a secondary structure along the length of the transcribed section of the RNA. The analysis of both primary or secondary structures requires the RNA end-labeling either at its 5' end using a kinase reaction with [gamma-32P]ATP, or at its 3' end using an RNA ligation reaction with [32P]pCp. End-labeled RNAs are then gradually breakdown using hydrolysing chemicals or a variety of enzymes targeting specific RNA sequences and secondary structure. The most commonly used enzymes are RNase A, T1, and V1. The partial digestion of the RNA reveals a mix of truncated RNA fragments of different lengths, called RNA ladder. The products are then separates through a high resolution gel system and subjected to autoradiographic analysis. Each visible fragment is labeled at one end, but comprises an enzyme specific sequence at the other end. Final comparison of the detected RNA ladders reveals a hypothetical model of the secondary RNA structure under assay conditions.