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

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.