title: Toward a general approach for RNA-templated hierarchical assembly of split-proteins.
abstract: The ability to conditionally turn on a signal or induce a function in the presence of a user-defined RNA target has potential applications in medicine and synthetic biology. Although sequence-specific pumilio repeat proteins can target a limited set of ssRNA sequences, there are no general methods for targeting ssRNA with designed proteins. As a first step toward RNA recognition, we utilized the RNA binding domain of argonaute, implicated in RNA interference, for specifically targeting generic 2-nucleotide, 3' overhangs of any dsRNA. We tested the reassembly of a split-luciferase enzyme guided by argonaute-mediated recognition of newly generated nucleotide overhangs when ssRNA is targeted by a designed complementary guide sequence. This approach was successful when argonaute was utilized in conjunction with a pumilio repeat and expanded the scope of potential ssRNA targets. However, targeting any desired ssRNA remained elusive as two argonaute domains provided minimal reassembled split-luciferase. We next designed and tested a second hierarchical assembly, wherein ssDNA guides are appended to DNA hairpins that serve as a scaffold for high affinity zinc fingers attached to split-luciferase. In the presence of a ssRNA target containing adjacent sequences complementary to the guides, the hairpins are brought into proximity, allowing for zinc finger binding and concomitant reassembly of the fragmented luciferase. The scope of this new approach was validated by specifically targeting RNA encoding VEGF, hDM2, and HER2. These approaches provide potentially general design paradigms for the conditional reassembly of fragmented proteins in the presence of any desired ssRNA target.
authors.affiliation: Department of Chemistry & Biochemistry, University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85721, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Furman
authors.forename: Ahmed H
authors.initials: AH
authors.lastname: Badran
authors.forename: Oluyomi
authors.initials: O
authors.lastname: Ajulo
authors.forename: Jason R
authors.initials: JR
authors.lastname: Porter
authors.forename: Cliff I
authors.initials: CI
authors.lastname: Stains
authors.forename: David J
authors.initials: DJ
authors.lastname: Segal
authors.forename: Indraneel
authors.initials: I
authors.lastname: Ghosh
mesh_terms: D004247:DNA / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000235:genetics; D042442:Vascular Endothelial Growth Factors / Q000737:chemistry / Q000235:genetics
chemical_list: D004247:DNA; D008156:Luciferases; D051736:Proto-Oncogene Proteins c-mdm2; D012313:RNA; D018719:Erb-b2 Receptor Tyrosine Kinases; D042442:Vascular Endothelial Growth Factors; C497206:MDM2 protein, human; C508053:ERBB2 protein, human