Versatile synthesis and rational design of caged morpholinos

Xiaohu Ouyang, Ilya A. Shestopalov, Surajit Sinha, Genhua Zheng, Cameron L W Pitt, Wen Hong Li, Andrew J. Olson, James K. Chen

Research output: Contribution to journalArticle

67 Citations (Scopus)

Abstract

Embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner, and deciphering these molecular mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision. Chemical technologies can enable such genetic manipulations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control. Here we report optimized methods for the design and synthesis of hairpin cMOs incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from commercially available reagents. Using this simplified procedure, we have systematically prepared cMOs with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successfully applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prepare a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chemical probes in vivo for functional genomic studies.

Original languageEnglish (US)
Pages (from-to)13255-13269
Number of pages15
JournalJournal of the American Chemical Society
Volume131
Issue number37
DOIs
StatePublished - Sep 23 2009

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Morpholinos
Genes
Oligonucleotides
Technology
Developmental Genes
Thermodynamic properties
Photons
Zebrafish
Thermodynamics
Embryonic Development

ASJC Scopus subject areas

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

Cite this

Ouyang, X., Shestopalov, I. A., Sinha, S., Zheng, G., Pitt, C. L. W., Li, W. H., ... Chen, J. K. (2009). Versatile synthesis and rational design of caged morpholinos. Journal of the American Chemical Society, 131(37), 13255-13269. https://doi.org/10.1021/ja809933h

Versatile synthesis and rational design of caged morpholinos. / Ouyang, Xiaohu; Shestopalov, Ilya A.; Sinha, Surajit; Zheng, Genhua; Pitt, Cameron L W; Li, Wen Hong; Olson, Andrew J.; Chen, James K.

In: Journal of the American Chemical Society, Vol. 131, No. 37, 23.09.2009, p. 13255-13269.

Research output: Contribution to journalArticle

Ouyang, X, Shestopalov, IA, Sinha, S, Zheng, G, Pitt, CLW, Li, WH, Olson, AJ & Chen, JK 2009, 'Versatile synthesis and rational design of caged morpholinos', Journal of the American Chemical Society, vol. 131, no. 37, pp. 13255-13269. https://doi.org/10.1021/ja809933h
Ouyang X, Shestopalov IA, Sinha S, Zheng G, Pitt CLW, Li WH et al. Versatile synthesis and rational design of caged morpholinos. Journal of the American Chemical Society. 2009 Sep 23;131(37):13255-13269. https://doi.org/10.1021/ja809933h
Ouyang, Xiaohu ; Shestopalov, Ilya A. ; Sinha, Surajit ; Zheng, Genhua ; Pitt, Cameron L W ; Li, Wen Hong ; Olson, Andrew J. ; Chen, James K. / Versatile synthesis and rational design of caged morpholinos. In: Journal of the American Chemical Society. 2009 ; Vol. 131, No. 37. pp. 13255-13269.
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