Poster number 130 submitted by Megan Van Horn
A method for isolation of target circular RNAs through specific biotinylated-DNA probes
Megan L. Van Horn (Department of Chemistry, Carnegie Mellon University), Marta Rachwalak (Department of Chemistry, Carnegie Mellon University), Anna M. Kietrys (Department of Chemistry, Carnegie Mellon University)
Circular RNAs (circRNA) are a class of long, endogenous RNAs possessing a covalently closed continuous loop structure and are formed through a combination of precursor mRNA (pre-mRNA) linear splicing and backsplicing1. This creates a unique junction of the downstream exon’s 3’-end spliced to the upstream exon’s 5’-end. Circular RNAs have extended life span2 and versatile interaction with small RNAs3,4 and proteins, and are promising biomarkers for various diseases and pathologies.
While there is a growing number of methods that can be used to identify a particular circRNA within a given population, the majority of these methods are destructive or non-isolating. In other words, the circular RNA cannot be used in downstream applications or not without further processing. This includes various RNA-sequencing methods5, as well as 2D gel electrophoresis and gel traps, among others6. However, the ability to isolate a particular circRNA of interest in a non-destructive manner is desirable for various experimental applications, such as modification/labeling, RNA sequencing, or biochemical assays.
Our group is working on a novel experimental tool to isolate particular circular RNAs from total RNA content. From prior research, we are using two previously-identified circRNAs as targets for the initial round of testing: a mitochondrial circRNA originating from the COX3 coding region and a chromosome 19 circRNA originating from the MYH14 gene that is found to be expressed in the brain7. Synthetic fluorescently-tagged circRNA have been generated and validated. Biotinylated-DNA probes have been developed to specifically bind to the backsplice junction of a target circular RNA. The binding and pull-down efficiency of the biotinylated-DNA probes is being tested within the pool of synthetic circRNA and cellular ones. We believe that this method can be a useful tool for future studies utilizing populations of circular RNA.
1. Xiao, J, Cohen, IR, Lajtha, A, et al. Circular RNAs, Biogenesis and Functions 2018, Vol. 1
2. Piwecka M, Glažar P, Hernandez-Miranda LR, et al. Science 2017.
3. Hansen TB, Jensen TI, Clausen BH, et al. Nature 2013. doi:10.1038/nature11993
4. Memczak S, Jens M, Elefsinioti A, et al. Nature 2013. doi:10.1038/nature11928
5. Panda, AC et al. Nucleic Acids Res. 2017.
6. Jeck, WR, Sharpless, NE, Nature Biotechnology 2014.
7. Rybak-Wolf, A, Stottmeister, C, Glažar, P, et al. Molecular Cell 2015. doi:10.1016/j.molcel.2015.03.027
Keywords: circular RNA, DNA probe