RNA viruses caused the three major pandemics of the 20th century, influenza, polio, and HIV. Despite the central roles of RNA in essentially all diseases, the RNA structures that can potentially be therapeutic targets are largely unknown. The ultimate goal of this research is to predict reliably the structures of RNA molecules from their sequences by using knowledge of the interactions directing RNA folding. The results should also lead to rational design of therapeutics. The ability to predict RNA structure also facilitates interpretation of sequences determined by genome projects and other sequencing efforts. The foundation for structure prediction will be advanced by studies of the thermodynamic and structural properties of oligonucleotides. Particular emphasis will be placed on the sequence dependence of stability and local three dimensional structures of internal loops. Secondary structure prediction by free energy minimization will be augmented with constraints from NMR. The NMR assisted prediction of secondary structure (NAPSS) method uses two unassigned spectra to restrict folding space by identifying helixes of canonical base pairs. This allows determination of pseudoknots, which are difficult to predict with other methods. Pseudoknots usually have significant functions, and are thus potential therapeutic targets. An approach is proposed that restricts folding space further on the basis of NMR chemical shifts. NAPSS is also a first step in assigning resonances for 3D structure determination. To advance predictions of 3D structure, benchmarks will be developed to test force fields and computational methods. For example, (1) the complete solution structure of a new and novel internal loop motif discovered in the previous grant period will be determined by NMR, (2) a less stable structure in equilibrium with this novel structure will also be determined, and (3) structural aspects of single stranded oligoribonucleotide tetramers will be determined by NMR and compared to predictions from molecular dynamics simulations. The power of predictive methods will be tested by experimentally determining the secondary and 3D structures of regions of influenza RNA predicted to fold into stable secondary structures. Initial results indicate there is a conformational switch between a pseudoknot and hairpin that may regulate splicing. Microarrays of short oligonucleotides and small molecules will be used to discover compounds that target the discovered structures and could potentially serve as therapeutics for natural strains and any engineered for bioterrorism. These approaches provide the foundation for rapidly designing therapeutics once a genome has been sequenced.

Public Health Relevance

Most diseases are mediated through RNA, and some viruses have RNA genomes. The goal of this research is to be able to predict reliably the structure of an RNA from its sequence in order to provide a foundation for rational design of therapeutics targeting RNA. The results may also help fight bioterrorism because they potentially provide a rapid way to design therapeutics once a genome has been sequenced.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM022939-38
Application #
8676808
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter
Project Start
Project End
Budget Start
Budget End
Support Year
38
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Rochester
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Rochester
State
NY
Country
United States
Zip Code
14627
Lenartowicz, Elzbieta; Kesy, Julita; Ruszkowska, Agnieszka et al. (2016) Self-Folding of Naked Segment 8 Genomic RNA of Influenza A Virus. PLoS One 11:e0148281
Chen, Jonathan L; Kennedy, Scott D; Turner, Douglas H (2015) Structural features of a 3' splice site in influenza a. Biochemistry 54:3269-85
Priore, Salvatore F; Kauffmann, Andrew D; Baman, Jayson R et al. (2015) The Influenza A PB1-F2 and N40 Start Codons Are Contained within an RNA Pseudoknot. Biochemistry 54:3413-5
Condon, David E; Kennedy, Scott D; Mort, Brendan C et al. (2015) Stacking in RNA: NMR of Four Tetramers Benchmark Molecular Dynamics. J Chem Theory Comput 11:2729-2742
Chen, Jonathan L; Bellaousov, Stanislav; Tubbs, Jason D et al. (2015) Nuclear Magnetic Resonance-Assisted Prediction of Secondary Structure for RNA: Incorporation of Direction-Dependent Chemical Shift Constraints. Biochemistry 54:6769-82
Kierzek, Ryszard; Turner, Douglas H; Kierzek, Elzbieta (2015) Microarrays for identifying binding sites and probing structure of RNAs. Nucleic Acids Res 43:1-12
Sripakdeevong, Parin; Cevec, Mirko; Chang, Andrew T et al. (2014) Structure determination of noncanonical RNA motifs guided by ¹H NMR chemical shifts. Nat Methods 11:413-6
Jiang, Tian; Kennedy, Scott D; Moss, Walter N et al. (2014) Secondary structure of a conserved domain in an intron of influenza A M1 mRNA. Biochemistry 53:5236-48
Dela-Moss, Lumbini I; Moss, Walter N; Turner, Douglas H (2014) Identification of conserved RNA secondary structures at influenza B and C splice sites reveals similarities and differences between influenza A, B, and C. BMC Res Notes 7:22
Kierzek, Elzbieta; Malgowska, Magdalena; Lisowiec, Jolanta et al. (2014) The contribution of pseudouridine to stabilities and structure of RNAs. Nucleic Acids Res 42:3492-501

Showing the most recent 10 out of 153 publications