Chemical profiling of Inosine using a Click-Compatible Acrylamide and EndoV Protein Público
Dailey, Deanna (Spring 2021)
Abstract
RNA undergoes extensive post-transcriptional modifications that have a range of biological effects. Inosine is a ubiquitous modification that effects coding and non-coding functions in several types of RNA. Altered Franklin-Crick base-pairing leads to modified coding in mRNA and also changes splicing patterns. This modification participates in cellular stress and immune responses. However, the field is limited by the lack of sensitive and selective editing detection methods. Current methods use a Michael addition to introduce adducts for high throughput sequencing techniques, however these methods often cannot distinguish inosine apart from a guanosine base, within error of PCR. These methods are time-intensive and costly as well. Herein, we synthesized an acrylamide derivative with click chemistry functionalization for enrichment and detection of inosine. This provides a cheaper, quicker way to probe editing rates on certain RNA substrates. Additionally, protein identification of these edits is a promising new direction of A-to-I edit determination. Current researchers have had success utilizing eEndoV protein to recognize and enrich inosine-containing RNA. Herein, we further explore the use of EndoV, and other homologs to probe A-to-I editing sites.
While this technology has the power to personalize disease treatment, there are some ethical concerns that might arise from this type of research. We as a Global Health Community must ensure that everyone receives equal access to this technology regardless of race or ethnicity, location, or cost. Additionally, we have to safeguard against using RNA studies and genomic data to define certain groups as less healthy, weaker, or less deserving.
Table of Contents
Chapter 1: Introduction……………………………………………………………………….…...1
1.1:RNA A-to-I editing…………………………………………………………………....2
1.2: Detecting RNA edits: RNA sequencing……………………………………………...4
1.3: Detecting RNA edits: Chemical Labeling……………………………………………5
1.4:Detecting RNA edits: Protein Recognition……………………………………………8
Chapter 2: Methods and Materials …….…….……………………………………….………….11
2.1: HER1 RNA A vs HER1 RNA I Labeling…………………………………………...11
2.2: Sensitivity of EPhAA Labeling Procedure………………………………………….11
2.3: Mock HER1 RNA A Editing and Labeling…………………………………………12
2.4: Synthesizing and determination of Efficiency of new EPhAA……………………..13
2.5: pETM-41 Cloning…………………………………………………………………..14
Chapter 3: Results and Discussion ……...……………………………………………………….17
3.1 HER1 RNA A vs HER1 RNA I Labeling……………………………………………17
3.2: Sensitivity of EPhAA Labeling Procedure………………………………………….19
3.3: Mock HER1 RNA A Editing and Labeling…………………………………………19
3.4: Synthesizing and determination of Efficiency of new EPhAA……………………..20
3.5: pETM Cloning…...………………………………………………………………….21
Chapter 4: Ethics…………………………………………………………………………………23
4.1:Introduction to Ethics and why it is Important? ….……………….………………...23
4.2: How will Genetic Information be Used?....................................................................24
4.3: How is Research Involved in Equality and Equity? ………………………………..24
4.4: How is Medicine Involved in Equality and Equity? ………………………………..26
4.5: Okay, so what? Where do we go from here? ……………………………………….27
Chapter 5: Conclusion and Future Direction………………………………………….…………29
Chapter 6: Resources…...……………………………………….……………………………….30
6.1: Chemistry Portion…………………………………………………………………...30
6.2: Ethics Portion………………………………………………………………………..31
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