Toward a general approach for RNA-templated hierarchical assembly of split-proteins

Jennifer L. Furman, Ahmed H. Badran, Oluyomi Ajulo, Jason R. Porter, Cliff I. Stains, David J. Segal, Indraneel Ghosh

Research output: Contribution to journalArticle

26 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)11692-11701
Number of pages10
JournalJournal of the American Chemical Society
Volume132
Issue number33
DOIs
StatePublished - Aug 25 2010

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Luciferases
RNA
Proteins
Zinc Fingers
Nucleotides
Synthetic Biology
Zinc
RNA Interference
Vascular Endothelial Growth Factor A
Bioelectric potentials
Scaffolds
Medicine
DNA
Enzymes

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Furman, J. L., Badran, A. H., Ajulo, O., Porter, J. R., Stains, C. I., Segal, D. J., & Ghosh, I. (2010). Toward a general approach for RNA-templated hierarchical assembly of split-proteins. Journal of the American Chemical Society, 132(33), 11692-11701. https://doi.org/10.1021/ja104395b

Toward a general approach for RNA-templated hierarchical assembly of split-proteins. / Furman, Jennifer L.; Badran, Ahmed H.; Ajulo, Oluyomi; Porter, Jason R.; Stains, Cliff I.; Segal, David J.; Ghosh, Indraneel.

In: Journal of the American Chemical Society, Vol. 132, No. 33, 25.08.2010, p. 11692-11701.

Research output: Contribution to journalArticle

Furman, JL, Badran, AH, Ajulo, O, Porter, JR, Stains, CI, Segal, DJ & Ghosh, I 2010, 'Toward a general approach for RNA-templated hierarchical assembly of split-proteins', Journal of the American Chemical Society, vol. 132, no. 33, pp. 11692-11701. https://doi.org/10.1021/ja104395b
Furman JL, Badran AH, Ajulo O, Porter JR, Stains CI, Segal DJ et al. Toward a general approach for RNA-templated hierarchical assembly of split-proteins. Journal of the American Chemical Society. 2010 Aug 25;132(33):11692-11701. https://doi.org/10.1021/ja104395b
Furman, Jennifer L. ; Badran, Ahmed H. ; Ajulo, Oluyomi ; Porter, Jason R. ; Stains, Cliff I. ; Segal, David J. ; Ghosh, Indraneel. / Toward a general approach for RNA-templated hierarchical assembly of split-proteins. In: Journal of the American Chemical Society. 2010 ; Vol. 132, No. 33. pp. 11692-11701.
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