Presentation description

The importance of understanding the structure of RNA molecules extends into many fields such as pharmacology and molecular biology. Having reliable methods to efficiently determine both secondary and tertiary structure can have a significant impact. In RNA, which is utilized to perform a wide variety of functions, many of the in vivo adopted structures are uncharacterized. SHAPE-seq is one of the current chemical-based sequencing methods used to identify the secondary structure of RNA through next generation sequencing platforms. While effective, the current protocol relies heavily on Reverse transcriptase PCR (RT-PCR) to convert extracted RNA into complementary DNA (cDNA), which has the potential to cause changes to the original sequence. The aim of this project is to modify the SHAPE-seq method to allow for the sequencing of native RNA strands directly on the nanopore sequencing platform by utilizing a halogenation reaction that selects for solvent available cytosines. The adducts created induce a unique signal that affects the base-calling data obtained through nanopore sequencing. Analysis of the base-calling data allows for the determination of the presence and location of secondary structures in extracted RNA sequences based on a nucleotide's exposure to the solvent (i.e. unstructured). In the case of halogenated cytosines it was found that sequence context has a significant effect on the base calling data that is obtained through nanopore sequencing. As a result, there will be an increased level of complexity when attempting to identify secondary and tertiary structures of RNA molecules in the presence of halogenating conditions.