Synthesis of a Click-Compatible Phenylacrylamide to Probe A-to-I RNA Editing Open Access

Korn, Megan (Spring 2020)

Permanent URL: https://etd.library.emory.edu/concern/etds/cj82k827k?locale=en
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Abstract

Adenosine-to-inosine (A-to-I) editing is one of the most common modifications in the human transcriptome, however full range of its biological function has not been fully explored. A lack of cost-effective, simple, and quick tools to probe A-to-I editing has limited research into functions of A-to-I sites and the therapeutic potential of ADAR. Herein, a click-compatible phenylacrylamide was developed to probe in vitro ADAR interactions with RNA substrates. N-(4-ethylnylphenyl)acrylamide (EPhAA) was synthesized in one-step with 42% yield, and carried out 90% percent conversion of inosine ribonucleoside. Using copper click chemistry, a Cy5 moiety was covalently attached to EPhAA-labeled RNA oligonucleotides and percent labeling was quantified with a gel shift assay. In vitro editing of HER1—a known ADAR substrate—was carried out using a mutant E1008Q ADAR1, which edits at a quicker rate than wild type ADAR1. Using EPhAA, Cy5 was click-conjugated to edited HER1 transcripts and editing was quantified with a gel shift assay. EPhAA successfully demonstrated enhanced editing of E1008Q ADAR1 versus wild type ADAR1. This system offers a cheaper, simpler, and quicker way to quantify A-to-I editing, as compared to other chemical labeling methods such as radioactive labeling and ICE-seq. The demonstrated labeling protocol is an ideal system for characterizing relative editing rates of mutated ADARs or for exploring novel RNA substrates of ADAR.

Table of Contents

1. Introduction …………………………………………………………………………………..…….… 1

1.1 Epitranscriptomics ……………………………………………….........…………………………. 1

1.2 A-to-I Editing ………………………………………………………………………..........…........ 1

1.3 Biological Function of A-to-I Editing ………………………………………....………..……. 2

1.4 ADAR as a Therapeutic Agent …….…………………………………………….…........…..... 4

1.5 Methods for Detecting Inosine in the Epitranscriptome ………………………….….…. 5

2. Results and Discussion …….…………………………………………………………………….... 9

2.1 Acrylamide Derivative Reactivity Panel ……………………………………………….....…. 9

2.2 Synthesis of N-(4-ethylnylphenyl)acrylamide ………………………………....……........ 10

2.3 Ribonucleoside Labeling and HPLC Analysis ……………………………....................... 10

2.4 RNA Oligonucleotide Labeling and PAGE Shift Analysis ………………….................. 13

2.5 HER1 RNA A versus HER1 RNA I Labeling and PAGE Shift Analysis …................... 15

2.6 HER1 RNA I Labeling Sensitivity and PAGE Shift Analysis ……………………........... 16

2.7 Mock HER1 RNA A Editing and PAGE Shift Analysis …………………........................ 17

2.8 ADAR1 Editing of HER1 and PAGE Shift Analysis …………………............................ 18

3. Conclusions and Limitations …….…….……………………………………………....……….. 20

4. Materials and Methods …….……………………………..…………………………………...….. 22

References …….………………………………………………………………………………...……..... 32

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