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An ESI-MS method for characterization of native and modified oligonucleotides used for RNA interference and other biological applications

Nature Protocols volume 3, pages 351–356 (2008)Cite this article

Abstract

RNA interference (RNAi) has become a powerful tool for investigating gene function, and, in addition, shows potential for the development of therapeutic agents. RNAi can be triggered in a variety of eukaryotic cells using small interfering RNA (siRNA), their double-stranded precursors (double-stranded RNA) and short hairpin precursors (shRNA). Here, we describe a protocol for analyzing these RNAs and their modifications using electrospray ionization mass spectrometry (ESI-MS). This protocol involves the desalting of nucleic acids using ammonium acetate precipitation, followed by characterization using ESI-MS. This protocol has been chiefly used for analyzing siRNAs and their chemical modifications, but it has also been used and can be applied to the analysis of a wide range of native and modified oligonucleotides. This protocol provides accurate information on molecular weight for a range of nucleic acids and can be completed in less than a day.

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Figure 1: Examples of modified and unmodified siRNAs and dsRNAs examined with mass spectrometry using this protocol.
Figure 2: Reconstructed ESI mass spectrum of a GFP-targeting siRNA (sense strand, 5′-GCA GCA CGA CUU CUU CAA GdTdT-3′; antisense strand, 5′-CUU GAA GAA GUC GUG CUG CdTdT-3′) containing a 5′-phosphate on the antisense strand.
Figure 3: Reconstructed ESI mass spectrum of a GFP-targeting siRNA (sense strand, 5′-GCA GCA CGA CUU CUU CAA GdTdT-3′; antisense strand, 5′-CUU GAA GAA GUC GUG CUG CdTdT-3′) containing a 5′-antisense phosphate after modification with a photolabile DMNPE group, as shown in Figure 1a.
Figure 4: Reconstructed ESI mass spectrum of a 21 mer oligonucleotide (sequence, 5′-CUU GAA GAA GUC GUG CUG CdTdT-3′) containing a C6 amino linker on the 5′-end in the presence of the same 21 mer oligonucleotide containing only a phosphate modification on the 5′-end of a known molecular weight as an internal standard.
Figure 5: Raw ESI mass spectrum (before mass reconstruction) of a GFP-targeting siRNA (sense strand, 5′-GCA GCA CGA CUU CUU CAA GdTdT-3′; antisense strand, 5′-CUU GAA GAA GUC GUG CUG CdTdT-3′) containing a 5′-antisense phosphate after modification with a photolabile DMNPE group, as shown in Figure 1a.

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Acknowledgements

The support of the NIH is gratefully acknowledged. We thank Professor William Gutheil for curating the Q-trap instrument that was used to generate the data within this manuscript.

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  1. Division of Pharmaceutical Sciences, 5005 Rockhill Road, University of Missouri, Kansas City, 64110, Missouri, USA

    Samit Shah & Simon H Friedman

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  1. Samit Shah
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Correspondence to Simon H Friedman.

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Shah, S., Friedman, S. 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). https://doi.org/10.1038/nprot.2007.535

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