Optimization of nucleotides dephosphorylation for RNA structural characterization by tandem mass spectrometry hyphenated with separation methods

Highlights

• Optimization of two dephosphorylation protocols for nucleotides.

• Fully dephosphorylated nucleosides in 4 h with 7.5 U of alkaline phosphatase.

• Fully dephosphorylated nucleosides overnight with 3.0 U of alkaline phosphatase.

Abstract

As part of RNA characterization, the identification of post-transcriptional modifications can be performed using hyphenation of separation methods with mass spectrometry. To identify RNA modifications with those methods, a first total digestion followed by a dephosphorylation step are usually required to reduce RNA to nucleosides. Even though effective digestion and dephosphorylation are essential to avoid further complications in analysis and data interpretation, to our knowledge, no standard protocol is yet referenced in the literature. Therefore, the aim of this work is to optimize the dephosphorylation step using a total extract of transfer RNA (tRNA)¹ from B. taurus as a model and to determine and fix two protocols, leading to complete dephosphorylation, based on time and bacterial alkaline phosphatase (BAP)² consumptions. Capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) was used to estimate the dephosphorylation efficiency of both protocols on many canonical and modified nucleotides. For a timesaving protocol, we established that full dephosphorylation was obtained after a 4-hour incubation at 37 °C with 7.5 U of BAP for 1 µg of tRNA. And for a BAP-saving protocol, we established that full dephosphorylation was obtained 3.0 U of BAP after an overnight incubation at 37 °C. Both protocols are suitable for quantitative analyses as no loss of analytes is expected. Moreover, they can be widely used for all other RNA classes, including messenger RNA or ribosomal RNA.

Introduction

Over the past few years, RNA studies have shown a growing interest due to the huge potential of RNA in therapeutic strategies such as diagnostic [1], treatment [2] or vaccination [3]. Structural information, including the identification and location of post-transcriptional modifications, is required to characterize RNA. Nowadays, more than 150 modified nucleosides are described in the literature and referenced in databases like Modomics [4]. These modifications are found in all types of RNA and are of great interest due to their involvement in the RNA structural stability [5] and many biological functions [6] but also in several diseases [7] as diabetes [8] or cancers [9].

Many techniques have been developed to analyze RNA post-transcriptional modifications, including separative methods such as high-performance liquid chromatography (LC) [8], [10], [11], [12], [13], [14], [15], [16], [17], [18] or capillary electrophoresis (CE) [19] hyphenated to tandem mass spectrometry (MS/MS). Indeed, on one hand, these separative methods, which have complementary selectivity, enable the characterization of several modified nucleosides including isomers [19]. On the other hand, MS has become a powerful tool for the identification and structural characterization of biomolecules due to its sensitivity and specificity. Using those couplings, the identification of RNA modifications is commonly performed at the nucleosides level, while their localization is determined at the oligonucleotides level. Both, CE and LC, could have been used in this study. However, CE offers the possibility to study RNA at both levels, nucleosides and oligonucleotides, with the same method [19], contrary to LC-MS, while maintaining the advantages of isomeric separation.

In order to digest RNA into nucleosides, the protocols usually described in the literature are based on two steps. The first one is a total digestion with nuclease P1 [10], [19] or with a combination of nuclease P1 and phosphodiesterase I [11], [20], leading to nucleotides. The second step is a dephosphorylation with bacterial alkaline phosphatase (BAP) [10], [11], [19], [20], [21]. Effective digestion and dephosphorylation are essential to avoid further complications in analysis and data interpretation. In-solution, nuclease P1 seems to be quite efficient with a reported complete digestion of DNA in less than 2 h [22]. In 1990, Crain et al proposed a protocol for both steps leading to full digestion and dephosphorylation [20]. In 2014, Su et al, proposed an entire protocol, from the sample preparation to the data interpretation, for the quantitative analysis of RNA modifications by LC-MS. The first digestion step was performed with benzonase and phosphodiesterase I, and the dephosphorylation step with alkaline phosphatase [18]. In 2016, Thüring et al published a similar protocol using nuclease P1 instead of benzonase [17]. However, none of them are describing the optimization process of the dephosphorylation step to obtain fully dephosphorylated nucleosides. Moreover, since then, several studies used various protocols [11], [19], [21]. A kit is also commercialized by New England BioLabs enabling one-step digestion and dephosphorylation of RNA and DNA, however no information on its enzyme composition is available. Therefore, we consider that there is no standard protocol for the dephosphorylation of nucleotides. Moreover, to the best of our knowledge, analyte recovery rates were never discussed as no step may implies a loss of analytes, and performance studies have not always been carried out for each new protocol, leaving the possibility of partial dephosphorylation. Indeed, most of the studies are using Multiple Reaction Monitoring (MRM) detection [8], [11], [12], [13], [14], [16], [17], [18], [21], [23] as it is really useful to increase sensitivity. However, it is not appropriate for untargeted studies in which nucleotides are remaining in the sample due to poor dephosphorylation or when nucleoside modifications are still unknown.

In this work, we propose to optimize the dephosphorylation step using BAP to fix the conditions of two different protocols based on two classical imperatives: (i) economical with reduced BAP consumption or (ii) fast with a short dephosphorylation time.