Heavy atoms show the way for dsRNA assay

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  • Published: Mar 15, 2018
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Heavy atoms show the way for dsRNA assay

Hard to assay dsRNA in mixtures

Double-stranded RNA (dsRNA) is generally found in small amounts since in most organisms (apart from some viruses) RNA tends to be single-stranded. Double-stranded RNA has potential uses in medicine and as an insecticide (either applied topically or genetically engineered in plants), which are examples of RNA interference (RNAi). However, dsRNA is usually produced from modified bacteria, where it is produced as a mixture with other types of RNA, thus making accurate dsRNA assays difficult.

The University of Sheffield and Syngenta researchers produced small amounts of isotopically labelled dsRNA, either in vitro by transcription from DNA in the presence of guanosine triphosphate labelled with 13C and 15N, or else from an E. coli strain given 15NH4Cl as the nitrogen source in its growing medium. The resulting labelled dsRNA molecules were then used as standards; comparison of the relative amounts of labelled and unlabelled nitrogen by mass spectrometry was used to calculate the amount of dsRNA present in RNA mixtures.

RNA examined by ion pair RP HPLC and LC-MS

A 15N13C 401 base pair dsRNA standard was isolated from the in vitro transcription products, while a 15N 756 base pair dsRNA standard was isolated from the products from the modified E. coli strain. The purification of the two standards from other types of RNA involved adding a little of the enzyme RNase A, incubating for 10 minutes and then isolating intact dsRNA by solid-phase extraction.

The standards were examined by ion-pair reversed phase HPLC using an Agilent 1100 Series instrument and a Thermo Fisher ProSwift RP-1S column. Two buffers were used as solvents: buffer A was 0.1 M aqueous tetraethylammonium acetate at pH 7.0 (containing 0.01% acetonitrile), while buffer B was an aqueous solution of 0.1 M tetraethylammonium acetate containing 25% acetonitrile. The amount of buffer B was increased from 22% to 73% in a series of gradients, with a flow rate of 1 mL/min, a column temperature of 50 °C and UV detection. The purities of the heavy atom dsRNA standards were confirmed by this method, and UV extinction coefficients were determined for the purified standards.

Samples of crude dsRNA spiked with labelled dsRNA standards were digested into oligoribonucleotides (i.e. much smaller chains) over 1 hour using RNase A. The products were identified by LC-MS. HPLC was carried out using a U3000 instrument fitted with an Accucore C18 column (both from Thermo Fisher). Two buffer solutions were again used: here buffer A was an aqueous solution of 20 mM tetraethylammonium acetate and 80 mM 1,1,1,3,3,3-hexafluoro-2-propanol, while buffer B had the same concentration of additives but contained 50% acetonitrile. The proportion of buffer B was increased from 10 to 80%, with a flow rate of 0.1 mL/min and a column temperature of 30 °C.

Mass spectrometry used a Bruker maXis ultra-high-resolution time-of-flight (UHR-TOF) instrument. Mongo Oligo Mass Calculator software was used to calculate all possible monoisotopic oligoribonucleotide fragments; from these the masses of their heavy atom analogues could be found and the ratios between the normal and heavy isotopic species could then be determined. By adding a known amount of 15N dsRNA standard, the amount of dsRNA in a mixture derived from modified E. coli could be calculated, following enzyme digestion to give oligoribonucleotides and LC-MS. The addition of standard and subsequent quantification was carried out in triplicate, each run giving a similar result for dsRNA concentration. Consistent concentration results were obtained both for 765 base pair dsRNA and also for 401 base pair dsRNA, both produced in RNA mixtures from modified E. coli.

Isotope method used successfully for dsRNA assay

The use of purified heavy element standards in combination with LC-MS is an accurate way to assay dsRNA in RNA mixtures without the need for purification. However, much more work is needed before dsRNA methods are inexpensive enough to be used routinely in agriculture.

Related Links

Rapid Communications in Mass Spectrometry, 2018, 32, 590-596. Kung et al.. Quantification of dsRNA using stable isotope labeling dilution liquid chromatography mass spectrometry.

Journal of Chromatography A, 2017, 1484, 14-25. Nwokeoji et al.. Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry.

Wikipedia, RNA Interference

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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