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)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM022939-37A1
Application #
8500857
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
1979-03-01
Project End
2017-04-30
Budget Start
2013-07-01
Budget End
2014-04-30
Support Year
37
Fiscal Year
2013
Total Cost
$482,023
Indirect Cost
$161,725
Name
University of Rochester
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
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
Condon, David E; Yildirim, Ilyas; Kennedy, Scott D et al. (2014) Optimization of an AMBER force field for the artificial nucleic acid, LNA, and benchmarking with NMR of L(CAAU). J Phys Chem B 118:1216-28
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
Andronescu, Mirela; Condon, Anne; Turner, Douglas H et al. (2014) The determination of RNA folding nearest neighbor parameters. Methods Mol Biol 1097:45-70
Priore, Salvatore F; Kierzek, Elzbieta; Kierzek, Ryszard et al. (2013) Secondary structure of a conserved domain in the intron of influenza A NS1 mRNA. PLoS One 8:e70615
Moss, Walter N (2013) Computational prediction of RNA secondary structure. Methods Enzymol 530:3-65
Priore, Salvatore F; Moss, Walter N; Turner, Douglas H (2013) Influenza B virus has global ordered RNA structure in (+) and (-) strands but relatively less stable predicted RNA folding free energy than allowed by the encoded protein sequence. BMC Res Notes 6:330
Moss, Walter N; Priore, Salvatore F; Turner, Douglas H (2011) Identification of potential conserved RNA secondary structure throughout influenza A coding regions. RNA 17:991-1011

Showing the most recent 10 out of 135 publications