Templated 3' terminal fluorescent labeling of RNA using Klenow DNA polymerase.

A long-standing challenge in the study of RNA structure-function dynamics using fluorescence-based methods has been the precise attachment of fluorophores to structured RNA molecules. Despite significant advancements in the field, existing techniques have limitations, especially for 3' end labeling of long, structured RNAs. In response to this challenge, we developed a chemo-enzymatic method that uses Klenow DNA polymerase to label RNAs.

A Newly Identified Peripheral Duplex Anchors and Stabilizes the MALAT1 Triplex.

The accumulation of the 8-kb oncogenic long noncoding MALAT1 RNA in cells is dependent on the presence of a protective triple helix structure at the 3' terminus. While recent studies have examined the functional importance of numerous base triples within the triplex and its immediately adjacent base pairs, the functional importance of peripheral duplex elements has not been thoroughly investigated. To investigate the functional importance of a peripheral linker region that was previously described as unstructured, we employed a variety of assays including thermal melting, protection from exonucleolytic degradation by RNase R, small-angle X-ray scattering, biochemical ligation and binding assays, and computational modeling.