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The synthesis of tetra-modified RNA for the multidimensional control of gene expression via light-activated RNA interference

Nature Protocols volume 9, pages 11–20 (2014)Cite this article

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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.

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Figure 1: Introduction of four photocleavable DMNPE groups into siRNA/dsRNA.
Figure 2: HPLC trace of siRNA duplex starting material before modification reaction, at both 260 and 346 nm.
Figure 3: HPLC trace of crude reaction mixture after reaction of siRNA and diazo-DMNPE.
Figure 4: HPLC trace of purified fraction 1 at 260 and 346 nm.
Figure 5: ESI mass spectrum of fraction 1, target tetra-modified RNA.
Figure 6: ESI mass spectrum of fraction 2, representative impurity.
Figure 7: Typical LARI result.

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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).

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Authors and Affiliations

  1. Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri, Kansas City, Missouri, USA

    Ashish Kala, Piyush K Jain, Dipu Karunakaran, Samit Shah & Simon H Friedman

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  1. Ashish Kala
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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.

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Correspondence to Simon H Friedman.

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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

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