RNA STRUCTURAL-DYNAMICAL HIERARCHY: SARS-COV, SARS-COV-2, AND DELTA/OMICRON VARIANT S2M MONOMERS, KISSING COMPLEXES, AND EXTENDED DUPLEXES

Author

Adam KensingerFollow

Defense Date

10-24-2024

Graduation Date

Fall 12-20-2024

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

School

School of Science and Engineering

Committee Chair

Jeffrey D. Evanseck

Committee Member

Michael Cascio

Committee Member

Mihaela-Rita Mihailescu

Committee Member

Ramkumar Rajamani

Keywords

RNA hairpin, functional-dynamics, COVID-19, antiviral strategy, structure-function relationships, structure prediction, entropy, bioinformatics, multivariate analysis, HIV-1 DIS

Abstract

The power and promise of ribonucleic acid (RNA) molecules are only beginning to be realized in living organisms and pharmaceutical intervention. Specifically, defining the chemical physics of RNA’s heterogenous structural ensemble and dynamics, which has experienced expansive growth over the last few years, is central to a complete understanding, yet continues to be a significant challenge. A 41-nucleotide hairpin called the stem-loop II motif (s2m), present in the viral genomes of severe acute respiratory syndrome (SARS), SARS-coronavirus-2 (SARS-CoV-2), and the Delta and Omicron variants responsible for the COVID-19 pandemic, is a potential antiviral target due to its high sequence conservation across several viral families. Despite the efforts of the community, data sets are still lacking to build a knowledge base of RNA structure and dynamics, and from a practical point of view, aid in the design of SARS-CoV-2 antiviral therapies. We leveraged microsecond timescale molecular dynamics (MD) simulations and other computational techniques with experimental data to address significant questions at the forefront of understanding the structural-dynamical hierarchy of RNA.

In our first effort, we predicted atomic coordinates for SARS-CoV-2 s2m consistent with NMR data and provided a comparative analysis with the X-ray crystal structure of SARS-CoV s2m. We found that increased flexibility and disorganization in the larger SARS-CoV-2 terminal nonaloop contributes to reduced kissing complex formation, while both s2m elements exhibit a similar L-shape due to distinct tertiary interactions (Chapter 2). In the second phase of our work, using our SARS-CoV-2 s2m model, we introduced the G15U mutation found in the Delta variant to understand the structural, energetic, and dynamical consequences of the single mutation. Profound consequences of the mutation were predicted to disrupt the L-shaped kink of SARS-CoV and SARS-CoV-2 s2m, resulting in a more linear hairpin, explaining the observed differences in electrophoretic migration and reduced homodimerization (Chapter 3). The next phase of our work followed the progression of the virus from the Delta to Omicron variant using bioinformatics analysis of BA.2* Omicron lineages, revealing a 26-nucleotide deletion in the s2m. The indel mutation removed the terminal loop which contained the mutually exclusive microRNA binding sites and kissing interaction palindrome sequence, potentially influencing viral fitness. Future research aims to determine whether the mutation provides a selective advantage and uncover the former role of the lost binding sites in the viral life cycle (Chapter 4). Finally, understanding the role of s2m in forming kissing complexes (KC) and extended duplexes (ED) in SARS-CoV, SARS-CoV-2, and Delta variants remains crucial. Using validated RNA structural prediction methods, we predicted the unreported 3D structures of SARS-CoV, SARS-CoV-2, and Delta s2m KC and ED, finding that SARS-CoV s2m forms stable stacked palindromic base triplets promoting a stable KC, consistent with experimental results showing stable SARS-CoV KC but mostly monomeric SARS-CoV-2 and Delta s2m (Chapter 5). Ultimately, this dissertation predicts s2m structures based on experimental data, uncovering dynamic differences arising from sequence mutations, and provides a foundation for future studies on the mechanisms of homodimerization, bridging the connection between structure and function within the s2m RNA structural-dynamical hierarchy of SARS-CoV, SARS-CoV-2, and the Delta/Omicron variants.

Language

English

Recommended Citation

Kensinger, A. (2024). RNA STRUCTURAL-DYNAMICAL HIERARCHY: SARS-COV, SARS-COV-2, AND DELTA/OMICRON VARIANT S2M MONOMERS, KISSING COMPLEXES, AND EXTENDED DUPLEXES (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/2399

Additional Citations

Figure 1: Reprinted with permission from Nucleic Acids Res. 2022, 50, 2, 1017-1032. Copyright 2022 Oxford University Press.

Figure 2: Reprinted with permission from RNA. 2023, 29, 11, 1754-1771. Copyright 2023 Cold Spring Harbor Publications under Creative Commons Attribution-NonCommercial 4.0 International License.

Three chapters of this dissertation are reprinted with permission from the American Chemical Society or Wiley Periodicals LLC.

Chapter 2: Reprinted with permission from ACS Phys. Chem Au 2023, 3, 1, 30–43. Copyright 2022 American Chemical Society. ACS Articles on Request author-directed link: http://pubs.acs.org/articlesonrequest/AOR-72PVZIBCR5J5MRU8BJ6F

Chapter 3: Reprinted with permission from ACS Phys. Chem Au 2023, 3, 5, 434–443. Copyright 2023 American Chemical Society. ACS Articles on Request author-directed link: http://pubs.acs.org/articlesonrequest/AOR-PKAP6SGDENXETDDGGXI8

Chapter 4: Reprinted with permission from J Med Virol. 2023, 95, e28141. Copyright 2022 Wiley Periodicals LLC.

Each of the three reprinted chapters begins with a journal cover graphic illustrated by Adam H. Kensinger and reprinted with permission from the American Chemical Society or Wiley Periodicals LLC.