Poster abstracts

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)

Abstract:
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.

References:
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