The RPL Effect
The Challenge of RNA Structure
Elucidating the structure of microscopic macromolecules is a significant challenge in the field of biology. Currently, most structural predictions rely on computer models, which are based on statistical methods and are not always accurate due to the lack of empirical evidence. Other methods for RNA structural studies include crystallography, electron microscopy, and spectroscopy, which are complex and difficult to scale.
One important aspect of RNA secondary structure determination concerns base pairing and the combinatorial structures formed when RNA complexes come into proximity with one another. These interactions form the basis for many of the catalytic and regulatory actions of ribozymes. Moreover, it is essential to understand these structures in detail to get a clearer picture of how these molecules operate.
RPL, aka “Ripple”
Researchers have developed a more straightforward method of decoding these secondary structures through a process called RNA proximity ligation (RPL, aka “ripple”). A manuscript published by Ramani et al. in Nature Biotechnology outlines the details of this RPL method. In short, this method enzymatically digests and ligates RNA molecules in situ, then generates libraries of chimeric RNA molecules which can form intra-molecular loops. These loops can then be sequenced to retrieve data used to determine RNA secondary structure based on pairwise contact maps.
Ramani et al. used this RPL method to determine the structure of yeast rRNA and eukaryotic snoRNAs in the spliceosome. Prior to performing the RPL ligation, the researchers had to extract the total RNA from yeast by first lysing and storing the cells in TRIzol, followed by purification with the Direct-zol RNA kit. After total RNA isolation, samples were digested with RNases and RNA Ligase was used to perform the RPL ligation. The resulting RPL ligations were then subjected to Illumina sequencing.
Their results led to a highly accurate, global determination of the RNA secondary structure through pairwise interaction. The authors further predict that when combined with current structure determination technologies, this method has the power to enable the high-throughput determination of secondary and tertiary RNA structure. To see their results and read more about RPL, you can read the paper here.
1. Ramani, V., Qiu, R., Shendure, J. 2015. High-throughput determination of RNA structure by proximity ligation. Nature Biotechnology 33(9): 980-984.