Title:
Experiments to optimize the ribose-seq protocol
Experiments to optimize the ribose-seq protocol
Author(s)
Bahl, Smriti
Advisor(s)
Storici, Francesca
Editor(s)
Collections
Supplementary to
Permanent Link
Abstract
Ribonucleoside monophosphates (rNMPs), the units of RNA, are the most abundant non-standard nucleotides found in genomic DNA. They can be incorporated by DNA polymerases during DNA replication and repair, by hydroxyl radicals during oxidative stress or during incomplete maturation of Okazaki fragments. rNMPs have profound consequences on genome stability, DNA structure, function, and various cellular processes. To better understand these effects, the Storici lab developed the ribose-seq protocol which is a systematic technique for capturing and analyzing rNMPs in genomic DNA. The aim of this study is to optimize the ribose-seq protocol by enhancing the efficiency and accuracy of rNMP detection while minimizing the required amount of starting DNA, thereby enabling easier acquisition also for possible clinical applications. We systematically investigated three key steps of the protocol: (1) adaptor ligation, (2) self-ligation using Arabidopsis thaliana tRNA ligase (AtRNL), and (3) degradation of linear single-stranded DNA (ssDNA) using exonuclease. Through rigorous experimentation and analysis, we observed that modifying the adaptor ligation conditions resulted in approximately a 30% increase in ligation efficiency of the adaptor to the fragmented DNA. The use of AtRNL with an extended incubation period at lower temperature enabled improved circularization of DNA containing the rNMPs, resulting in more abundant ribose-seq library product. Furthermore, novel exonucleases were evaluated as potential replacements for T5 exonuclease in order to effectively eliminate the remaining linear ssDNA following AtRNL self-ligation and protect the circular ssDNA structures containing rNMPs from exonuclease-mediated degradation. To validate the findings of this project, ribose-seq libraries were constructed using Saccharomyces cerevisiae DNA, demonstrating the potential to reduce the starting DNA amount by up to 50%. These findings present a significant advancement in the ribose-seq methodology, enabling researchers to investigate ribonucleotide-mediated genomic processes with enhanced sensitivity and reduced resource requirements.
Sponsor
Date Issued
2023-07-31
Extent
Resource Type
Text
Resource Subtype
Thesis