Viral RNA, like the human transcriptome, is punctuated by infrequent but critical base modifications and non- Watson-Crick motifs. Many knowledge gaps exist in understanding where, when and why certain modifications such as pseudouridine (?) and N6-methyl-adenosine (m6A) are enzymatically written onto mRNA. Similarly, guanosine-rich regions of viral RNA and the human transcriptome that may potentially fold to G-quadruplex motifs are conserved in regulatory regions controlling translation and viral replication for reasons that remain unclear. This research project hypothesizes that secondary structural motifs such as stem-loop structures and G-quadruplexes constitute the recognition sites for RNA modification. Additionally, these sites are hotspots for oxidative modification (8-oxo-7,8-dihydroguanosine, rOG) such as occurs during oxidative stress generated by viral infections. Thus, the project will examine the interplay of base modification (pseudouridinylation, guanosine oxidation and adenosine methylation) with secondary structural motifs in RNA. New innovative chemical biology tools will be developed to sequence long mRNA strands for folded structures by examining the ability of protein nanopores to thread and translocate folded or unfolded RNA. Similarly, base modifications will be identified using specific chemistries to amplify signals from base modification.
The specific aims are to (1) investigate the sequence vs. structural motif of pseudouridine locations in ZIKV RNA, (2) sequence for rOG and correlate sites with secondary structure vs. solvent exposure, and (3) correlate G4 folds of ZIKV RNA with m6A. The human health relevance of this research is to provide foundational science for understanding the molecular choreography of mRNA, both human and viral, in order to advance health strategies combatting viral infection, cancer, and age-related disorders.

Public Health Relevance

The proposed research is relevant to public health because it will provide methods for sequencing viral genomes and human transcriptomes for RNA modifications, some of which may be induced by oxidative stress or viral infection; these modifications will then be correlated to non-canonical structural motifs in nucleic acids. Oxidative stress is a causative agent in age- related disorders including cancer, diabetes, and Alzheimer?s disease, as well as playing a role in injury and inflammation-related disorders such as traumatic brain injury (TBI), and methylation markers in RNA appear to modulate viral genome replication and expression. The project is therefore relevant to NIH?s mission to understand RNA chemistry and its underlying roles in disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM093099-09A1
Application #
10051192
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Sakalian, Michael
Project Start
2011-09-01
Project End
2024-05-31
Budget Start
2020-09-01
Budget End
2021-05-31
Support Year
9
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Utah
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Zeng, Tao; Fleming, Aaron M; Ding, Yun et al. (2018) Nanopore Analysis of the 5-Guanidinohydantoin to Iminoallantoin Isomerization in Duplex DNA. J Org Chem 83:3973-3978
Edwards, M A; Robinson, D A; Ren, H et al. (2018) Nanoscale electrochemical kinetics & dynamics: the challenges and opportunities of single-entity measurements. Faraday Discuss 210:9-28
Tan, Cherie S; Fleming, Aaron M; Ren, Hang et al. (2018) ?-Hemolysin Nanopore Is Sensitive to Guanine-to-Inosine Substitutions in Double-Stranded DNA at the Single-Molecule Level. J Am Chem Soc 140:14224-14234
Ren, Hang; Cheyne, Cameron G; Fleming, Aaron M et al. (2018) Single-Molecule Titration in a Protein Nanoreactor Reveals the Protonation/Deprotonation Mechanism of a C:C Mismatch in DNA. J Am Chem Soc 140:5153-5160
Fleming, Aaron M; Ding, Yun; Burrows, Cynthia J (2017) Sequencing DNA for the Oxidatively Modified Base 8-Oxo-7,8-Dihydroguanine. Methods Enzymol 591:187-210
Zeng, Tao; Fleming, Aaron M; Ding, Yun et al. (2017) Interrogation of Base Pairing of the Spiroiminodihydantoin Diastereomers Using the ?-Hemolysin Latch. Biochemistry 56:1596-1603
Alenko, Anton; Fleming, Aaron M; Burrows, Cynthia J (2017) Reverse Transcription Past Products of Guanine Oxidation in RNA Leads to Insertion of A and C opposite 8-Oxo-7,8-dihydroguanine and A and G opposite 5-Guanidinohydantoin and Spiroiminodihydantoin Diastereomers. Biochemistry 56:5053-5064
Johnson, Robert P; Fleming, Aaron M; Perera, Rukshan T et al. (2017) Dynamics of a DNA Mismatch Site Held in Confinement Discriminate Epigenetic Modifications of Cytosine. J Am Chem Soc 139:2750-2756
An, Na; Fleming, Aaron M; Burrows, Cynthia J (2016) Human Telomere G-Quadruplexes with Five Repeats Accommodate 8-Oxo-7,8-dihydroguanine by Looping out the DNA Damage. ACS Chem Biol 11:500-7
Tan, Cherie S; Riedl, Jan; Fleming, Aaron M et al. (2016) Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured Using the ?-Hemolysin Nanopore. ACS Nano 10:11127-11135

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