PhD defense: Łukasz Jan Kiełpiński

High-throughput sequencing based methods of RNA structure investigation

Supervisors:
Associate Professor Jan Christiansen and Associate Professor Jeppe Vinther

Examination committee:
Prof. Peter Brodersen, University of Copenhagen (chair)
Prof. Jørgen Kjems, University of Aarhus
Prof. Eric Westhof, University of Strasbourg

Abstract:
In this thesis we describe the development of four related methods for RNA structure probing that utilize massive parallel sequencing. Using them, we were able to gather structural data for multiple, long molecules simultaneously. First, we have established an easy to follow experimental and computational protocol for detecting the reverse transcription termination sites (RTTS-Seq). This protocol was subsequently applied to hydroxyl radical footprinting of three dimensional RNA structures to give a probing signal that correlates well with the RNA backbone solvent accessibility. Moreover, we applied RTTS-Seq to detect antisense oligonucleotide binding sites within a transcriptome. In this case, we applied an enrichment strategy to greatly reduce the background. Finally, we have modified the RTTS-Seq to study the secondary structure of 3’ untranslated regions. In the course of this thesis we describe several computational methods. One that alleviates PCR bias by estimating number of unique molecules existing before the amplification, and two methods for data normalization: one applicable when the paired end sequencing is performed, and the other that works with the single read sequencing with known priming sites.