Abstract
Light-activated RNA interference (LARI) is an effective way to control gene expression with light. This, in turn, allows for the spacing, timing and degree of gene expression to be controlled by the spacing, timing and amount of light irradiation. The key mediators of this process are siRNA or dsRNA that have been modified with four photocleavable groups of dimethoxy nitro phenyl ethyl (DMNPE), located on the four terminal phosphate groups of the duplex RNA. These mediators can be easily synthesized and purified using two readily available products: synthetic RNA oligonucleotides and DMNPE-hydrazone. The synthesis of the tetra-DMNPE–modified duplex RNA is made possible by a remarkable regiospecificity of DMNPE for terminal phosphates (over internal phosphates or nucleobases) that we have previously identified. The four installed DMNPE groups effectively limit RNAi until irradiation cleaves them, releasing native, active siRNA. By using the described protocol, any process that is mediated by RNAi can be controlled with light. Although other methods exist to control gene expression with light by using specialized reagents, this method requires only two commercially available products. The protocol takes ∼3 d in total for the preparation of modified RNA.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Jain, P.K., Shah, S. & Friedman, S.H. Patterning of gene expression using new photolabile groups applied to light activated RNAi. J. Am. Chem. Soc. 133, 440–446 (2010).
Kala, A. & Friedman, S.H. Enhanced light-activated RNA interference using phosphorothioate-based dsRNA precursors of siRNA. Pharm. Res. 28, 3050–3057 (2011).
Shah, S. & Friedman, S.H. Tolerance of RNA interference toward modifications of the 5′ antisense phosphate of small interfering RNA. Oligonucleotides 17, 35–43 (2007).
Shah, S., Jain, P.K., Kala, A., Karunakaran, D. & Friedman, S.H. Light-activated RNA interference using double-stranded siRNA precursors modified using a remarkable regiospecificity of diazo-based photolabile groups. Nucleic Acids Res. 37, 4508–4517 (2009).
Shah, S., Rangarajan, S. & Friedman, S.H. Light activated RNA interference. Angew. Chem. Int. Edit. 44, 1328–1332 (2005).
Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).
Elbashir, S.M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).
Kim, D.H. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23, 222–226 (2005).
Monroe, W.T., McQuain, M.M., Chang, M.S., Alexander, J.S. & Haselton, F.R. Targeting expression with light using caged DNA. J. Biol. Chem. 274, 20895–20900 (1999).
Furuta, T. et al. Brominated 7-hydroxycoumarin-4-ylmethyls: photolabile protecting groups with biologically useful cross-sections for two photon photolysis. Proc. Natl. Acad. Sci. USA 96, 1193–1200 (1999).
Nguyen, Q.N. et al. Light controllable siRNAs regulate gene suppression and phenotypes in cells. Biochim. Biophys. Acta 1758, 394–403 (2006).
Cruz, F.G., Koh, J.T. & Llink, K.H. Light-activated gene expression. J. Am. Chem. Soc. 122, 8777–8778 (2000).
Krock, L. & Heckel, A. Photoinduced transcription by using temporarily mismatched caged oligonucleotides. Angew. Chem. Int. Edit. 44, 471–473 (2005).
Young, D.D., Lusic, H., Lively, M.O., Yoder, J.A. & Deiters, A. Gene silencing in mammalian cells with light-activated antisense agents. Chembiochem 9, 2937–2940 (2008).
Shestopalov, I.A., Sinha, S. & Chen, J.K. Light-controlled gene silencing in zebrafish embryos. Nat. Chem. Biol. 3, 650–651 (2007).
Tang, X., Maegawa, S., Weinberg, E.S. & Dmochowski, I.J. Regulating gene expression in zebrafish embryos using light-activated, negatively charged peptide nucleic acids. J. Am. Chem. Soc. 129, 11000–11001 (2007).
Oliveira, S., Fretz, M.M., Hogset, A., Storm, G. & Schiffelers, R.M. Photochemical internalization enhances silencing of epidermal growth factor receptor through improved endosomal escape of siRNA. Biochim. Biophys. Acta 1768, 1211–1217 (2007).
Oliveira, S., van Rooy, I., Kranenburg, O., Storm, G. & Schiffelers, R.M. Fusogenic peptides enhance endosomal escape improving siRNA-induced silencing of oncogenes. Int. J. Pharm. 331, 211–214 (2007).
Boe, S., Longva, A.S. & Hovig, E. Photochemically induced gene silencing using small interfering RNA molecules in combination with lipid carriers. Oligonucleotides 17, 166–173 (2007).
Endoh, T., Sisido, M. & Ohtsuki, T. Cellular siRNA delivery mediated by a cell-permeant RNA-binding protein and photoinduced RNA interference. Bioconjug. Chem. 19, 1017–1024 (2008).
Braun, G.B. et al. Laser-activated gene silencing via gold nanoshell-siRNA conjugates. ACS Nano 3, 2007–2015 (2009).
Endoh, T., Sisido, M. & Ohtsuki, T. Spatial regulation of specific gene expression through photoactivation of RNAi. J. Control Release 137, 241–245 (2009).
Shah, S. & Friedman, S.H. An ESI-MS method for characterization of native and modified oligonucleotides used for RNA interference and other biological applications. Nat. Protoc. 3, 351–356 (2008).
Chiu, Y.L. & Rana, T.M. RNAi in human cells: basic structural and functional features of small interfering RNA. Mol. Cell 10, 549–561 (2002).
Acknowledgements
We thank W.G. Gutheil for guidance on mass spectrometry issues. We thank A.K. Mitra and D. Pal for guidance on cell culture issues. This work was supported by a grant from the US National Science Foundation (CHE-1052871).
Author information
Authors and Affiliations
Contributions
A.K., P.K.J., D.K., S.S. and S.H.F were instrumental in developing and refining the procedures described over many years, including synthesis, purification and analytical methods. A.K. prepared the specific samples that were used to illustrate the protocol and wrote the methods portion of the protocol. S.H.F. guided the work and wrote and edited the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Kala, A., Jain, P., Karunakaran, D. et al. The synthesis of tetra-modified RNA for the multidimensional control of gene expression via light-activated RNA interference. Nat Protoc 9, 11–20 (2014). https://doi.org/10.1038/nprot.2013.165
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2013.165
This article is cited by
-
Construction of a reduction-responsive oligonucleotide via a post-modification approach utilizing 4-nitrophenyl diazomethane
Polymer Journal (2021)
-
Optoribogenetic control of regulatory RNA molecules
Nature Communications (2020)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.