Photocaged Morpholino Oligomers for the Light-Regulation of Gene Function in Zebrafish and Xenopus Embryos

Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States.
Journal of the American Chemical Society (Impact Factor: 12.11). 10/2010; 132(44):15644-50. DOI: 10.1021/ja1053863
Source: PubMed


Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these reagents limits their utility for applications that require spatiotemporal control, such as tissue-specific gene disruptions in embryos. Here we report a novel and efficient synthetic route for incorporating photocaged monomeric building blocks directly into morpholino oligomers and demonstrate the utility of these caged morpholinos in the light-activated control of gene function in both cell culture and living embryos. We demonstrate that a caged morpholino that targets enhanced green fluorescent protein (EGFP) disrupts EGFP production only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio) and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin phenotype in a UV-dependent manner. Our results suggest that photocaged morpholinos are readily synthesized and highly efficacious tools for light-activated spatiotemporal control of gene expression in multiple contexts.

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Available from: Alexander Deiters
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    • "Similar results were also observed when caged circular antisense oligonucleotides PS2 and PS3 were applied. The knockdown of GFP expression was achieved through non-enzyme involved antisense strategy which is similar to literature report (48) of morpholino oligomers caged with multiple photolabile moieties. Moreover, bright-field images with Hoechst nuclei staining and light irradiation of HeLa cells confirmed the viability of the cells over the course of the experiments (Supplementary Figure S7 and S8). "
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    ABSTRACT: We synthesized three 20mer caged circular antisense oligodeoxynucleotides (R20, R20B2 and R20B4) with a photocleavable linker and an amide bond linker between two 10mer oligodeoxynucleotides. With these caged circular antisense oligodeoxynucleotides, RNA-binding affinity and its digestion by ribonuclease H were readily photomodulated. RNA cleavage rates were upregulated ∼43-, 25- and 15-fold for R20, R20B2 and R20B4, respectively, upon light activation in vitro. R20B2 and R20B4 with 2- or 4-nt gaps in the target RNA lost their ability to bind the target RNA even though a small amount of RNA digestion was still observed. The loss of binding ability indicated promising gene photoregulation through a non-enzymatic strategy. To test this strategy, three caged circular antisense oligonucleotides (PS1, PS2 and PS3) with 2′-OMe RNA and phosphorothioate modifications were synthesized to target GFP expression. Upon light activation, photomodulation of target hybridization and GFP expression in cells was successfully achieved with PS1, PS2 and PS3. These caged circular antisense oligonucleotides show promising applications of photomodulating gene expression through both ribonuclease H and non-enzyme involved antisense strategies.
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    • "However, just as with mutations, if a gene has both early and late functions, blocking the early function with MOs often precludes studying the late function. Several studies have improved upon this system by developing MOs whose functions can be initially blocked and then released upon UV illumination (Deiters et al., 2010; Shestopalov and Chen, 2010; Shestopalov et al., 2007; Tomasini et al., 2009), providing one type of temporal control over MO activity. We have taken this one step further and here provide evidence for temporal control of MO function by activation and inactivation of MOs using light application in developing zebrafish embryos. "
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    ABSTRACT: To understand the molecular mechanisms of development it is essential to be able to turn genes on and off at will and in a spatially restricted fashion. Morpholino oligonucleotides (MOs) are very common tools used in several model organisms with which it is possible to block gene expression. Recently developed photo-activated MOs allow control over the onset of MO activity. However, deactivation of photo-cleavable MO activity has remained elusive. Here, we describe photo-cleavable MOs with which it is possible to activate or de-activate MO function by UV exposure in a temporal and spatial manner. We show, using several different genes as examples, that it is possible to turn gene expression on or off both in the entire zebrafish embryo and in single cells. We use these tools to demonstrate the sufficiency of no tail expression as late as tailbud stage to drive medial precursor cells towards the notochord cell fate. As a broader approach for the use of photo-cleavable MOs, we show temporal control over gal4 function, which has many potential applications in multiple transgenic lines. We demonstrate temporal manipulation of Gal4 transgene expression in only primary motoneurons and not secondary motoneurons, heretofore impossible with conventional transgenic approaches. In another example, we follow and analyze neural crest cells that regained sox10 function after deactivation of a photo-cleavable sox10-MO at different time points. Our results suggest that sox10 function might not be critical during neural crest formation.
    Full-text · Article · May 2012 · Development
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    • "NPOM could then be simply removed by UV irradiation (at 365 nM). As a proof of principle, they showed that they could readily target expression of green fluorescent protein (expressed as an mRNA) in a light-sensitive manner (Deiters et al., 2010). "
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    ABSTRACT: Chemical genetics, or chemical biology, has become an increasingly powerful method for studying biological processes. The main objective of chemical genetics is the identification and use of small molecules that act directly on proteins, allowing rapid and reversible control of activity. These compounds are extremely powerful tools for researchers, particularly in biological systems that are not amenable to genetic methods. In addition, identification of small molecule interactions is an important step in the drug discovery process. Increasingly, the African frog Xenopus is being used for chemical genetic approaches. Here, we highlight the advantages of Xenopus as a first-line in vivo model for chemical screening as well as for testing reverse engineering approaches.
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