title: Computational prediction and biochemical characterization of novel RNA aptamers to Rift Valley fever virus nucleocapsid protein.
abstract: Rift Valley fever virus (RVFV) is a potent human and livestock pathogen endemic to sub-Saharan Africa and the Arabian Peninsula that has potential to spread to other parts of the world. Although there is no proven effective and safe treatment for RVFV infections, a potential therapeutic target is the virally encoded nucleocapsid protein (N). During the course of infection, N binds to viral RNA, and perturbation of this interaction can inhibit viral replication. To gain insight into how N recognizes viral RNA specifically, we designed an algorithm that uses a distance matrix and multidimensional scaling to compare the predicted secondary structures of known N-binding RNAs, or aptamers, that were isolated and characterized in previous in vitro evolution experiment. These aptamers did not exhibit overt sequence or predicted structure similarity, so we employed bioinformatic methods to propose novel aptamers based on analysis and clustering of secondary structures. We screened and scored the predicted secondary structures of novel randomly generated RNA sequences in silico and selected several of these putative N-binding RNAs whose secondary structures were similar to those of known N-binding RNAs. We found that overall the in silico generated RNA sequences bound well to N in vitro. Furthermore, introduction of these RNAs into cells prior to infection with RVFV inhibited viral replication in cell culture. This proof of concept study demonstrates how the predictive power of bioinformatics and the empirical power of biochemistry can be jointly harnessed to discover, synthesize, and test new RNA sequences that bind tightly to RVFV N protein. The approach would be easily generalizable to other applications.
authors.affiliation: Division of Biological Sciences, The University of Montana, Missoula, Montana 59812, USA.
authors.forename: Mary
authors.initials: M
authors.lastname: Ellenbecker
authors.affiliation: Division of Biological Sciences, The University of Montana, Missoula, Montana 59812, USA.
authors.forename: Jeremy
authors.initials: J
authors.lastname: St Goddard
authors.affiliation: Department of Computer Science, The University of Montana, Missoula, Montana 59812, USA; Department of Chemistry and Biochemistry, The University of Montana, Missoula, Montana 59812, USA.
authors.forename: Alec
authors.initials: A
authors.lastname: Sundet
authors.affiliation: Division of Biological Sciences, The University of Montana, Missoula, Montana 59812, USA.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Lanchy
authors.affiliation: Department of Computer Science, The University of Montana, Missoula, Montana 59812, USA; Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, Montana 59812, USA.
authors.forename: Douglas
authors.initials: D
authors.lastname: Raiford
authors.affiliation: Division of Biological Sciences, The University of Montana, Missoula, Montana 59812, USA; Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, Montana 59812, USA. Electronic address: stephen.lodmell@umontana.edu.
authors.forename: J Stephen
authors.initials: JS
authors.lastname: Lodmell
mesh_terms: D000465:Algorithms; D052157:Aptamers, Nucleotide; D001483:Base Sequence; D057809:HEK293 Cells; D006801:Humans; D049410:Luciferases, Renilla / Q000235:genetics; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012295:Rift Valley Fever; D012296:Rift Valley fever virus / Q000235:genetics / Q000502:physiology; D014779:Virus Replication
chemical_list: D052157:Aptamers, Nucleotide; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D049410:Luciferases, Renilla
keywords: Aptamers; Nucleocapsid protein; RNA structure prediction; Rift Valley fever virus; Viral inhibition