Nucleic Acid Structure Determination - In-line Probing

In-line Probing

In-line probing does not involve treatment with any type of chemicals or reagents to modify exposed RNA structures. This type of probing assay uses the structure dependent cleavage of RNA, as areas that are single stranded are more flexible and over time will degrade, as RNA structure is not always stable. The process of in-line probing is often used to determine changes in structure due to ligand binding as this can result in different cleavage patterns. The process of in-line probing involves incubation of structural or functional RNAs over a long period of time, can be several days, but varies in each experiment, and then running the incubated products on a gel to visualize the bands. This experiment is often done using two different conditions: 1) with ligand and 2) in the absence of ligand. Cleavage results in shorter band lengths and is indicative of areas that are not basepaired, as those that are tend to be less sensitive to spontaneous cleavage. In line probing is a functional assay in ligand binding changes of RNA because it can show directly the change in flexibility and binding of regions of DNA in response to a ligand, as well as compare that response to analogous ligands. An in-line probing assay is then commonly used in dynamic studies, specifically when examining riboswitches