Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

Summary

MALDI-TOF was used to characterize fragments obtained from the reactivity between oxidized RNA and the exoribonuclease Xrn-1. The present protocol describes a methodology that can be applied to other processes involving RNA and/or DNA.

Abstract

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, and free radicals, are ubiquitous. As a result, RNA undergoes various reactions that include its cleavage, degradation, or modification, leading to biologically relevant species with distinct functions and implications. One example is the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG), which may occur in the presence of reactive oxygen species (ROS). Overall, procedures that characterize such products and transformations are largely valuable to the scientific community. To this end, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is a widely used method. The present protocol describes how to characterize RNA fragments formed after enzymatic treatment. The chosen model uses a reaction between RNA and the exoribonuclease Xrn-1, where enzymatic digestion is halted at oxidized sites. Two 20-nucleotide long RNA sequences [5'-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5'-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU] were obtained via solid-phase synthesis, quantified by UV-vis spectroscopy, and characterized via MALDI-TOF. The obtained strands were then (1) 5'-phosphorylated and characterized via MALDI-TOF; (2) treated with Xrn-1; (3) filtered and desalted; (4) analyzed via MALDI-TOF. This experimental setup led to the unequivocal identification of the fragments associated with the stalling of Xrn-1: [5'-H₂PO₄-(8-oxoG)G CUA AAA GU], [5'-H₂PO₄-(8-oxoG)(8-oxoG) CUA AAA GU], and [5'-H₂PO₄-(8-oxoG) CUA AAA GU]. The described experiments were carried out with 200 picomols of RNA (20 pmol used for MALDI analyses); however, lower amounts may result in detectable peaks with spectrometers using laser sources with more power than the one used in this work. Importantly, the described methodology can be generalized and potentially extended to product identification for other processes involving RNA and DNA, and may aid in the characterization/elucidation of other biochemical pathways.

Introduction

MALDI-TOF^1,2,3 is a widely used technique for the characterization and/or detection of molecules of varying sizes and characteristics. Some of its uses include diverse applications such as detecting tannins from natural resources⁴, imaging metabolites in food⁵, discovery or monitoring of cellular drug targets or markers⁶, and clinical diagnostics⁷, to name a few. Of relevance to the present work is the use of MALDI-TOF with DNA or RNA, with its use on oligonucleotides dating back t

Protocol

RNase-free ultra pure water (Table 1) was used for the present study.

1. Concentration determination of RNA solution

1. Prepare the RNA sample following the steps below.
   1. Use a microcentrifuge tube (0.6 mL) to prepare a solution of RNA by diluting 1 µL of stock solution (obtained via solid-phase synthesis)¹⁹ into 159 µL of RNase-free H₂O. Mix the solution by pipetting the mixture up and down repeatedly (10x).
      NOTE: Since a wide range of cuvettes are commercially available, the needed volumes may differ. Cuvettes with a 1 cm pa

Results

The oligonucleotides used in this work were synthesized, characterized, and quantified prior to use. The concentration of all oligonucleotides was determined via UV-vis spectroscopy recorded at 90 °C to avoid erroneous readings arising from the potential formation of the secondary structures. Figure 3 displays the spectra of the model oligonucleotides of RNA used in this work, taken at room temperature and after applying heat.

The overall procedure, ...

Discussion

The main challenge in this workflow arose between finalizing the experiments and carrying out the mass spectrometric analyses. Experiments were carried out and completed at the University of Colorado Denver and shipped (overnight) to the Colorado State University facilities. Data acquisition was carried out upon receipt, as per convenience. Several unexpected circumstances led to time delays in the process. In one instance, unexpected instrument malfunctions required the samples to be frozen (one time for 21 days) prior ...

Materials

Name	Company	Catalog Number	Comments
0.6 mL MCT Graduated Violet	Fisher Scientific	05-408-127
6’-Trihydroxyacetophenone monohydrate 98%	Sigma Aldrich	480-66-0
Acetonitrile 99.9%, HPLC grade	Fisher Scientific	75-05-8
Adenosine triphosphate, 10 mM	New Englang Bioscience	P0756S
Ammonium citrate, dibasic 98%	Sigma Aldrich	3012-65-5
Ammonium Fluoride 98.0%, ACS grade	Alfa Aesar	12125-01-8
Bruker bacterial test standard	Bruker Daltonics	8255343
Commercial source of Xrn-1	New England BioLabs	M0338S
Diethyl pyrocarbonate, 97%	ACROS Organics	A0368487
Flex analysis software	Bruker daltonics		FlexAnalysis software version 3.4, Bruker Daltonics
Lambda 365 UV-vis spectrophotometer	Perkin Elmer
MALDI plate: MSP 96 ground steel target	Bruker Daltonics	280799
Mass Spectrometer	Bruker		Microflex LRFTOF mass spectrometer (Bruker Daltonics, Billerica, MA)
Mili-Q IQ 7000	Milipore Sigma		A Mili-Q system was used to purify all water used in this work
Nanosep Centrifugal Devices with OmegaTM Membrane 10 K, blue (24/pkg)	Pall Corporation	OD010C33	filter media, Omega (modified polyethersulfone) 10 K pore size
NEBuffer 3	New England Biolabs	B7003S	This is solution B
Oligo Analyzer tool	IDT-DNA		https://www.idtdna.com/calc/analyzer
Pipette tips P10	Fisher Scientific	02-707-441
Pipette tips P200	Fisher Scientific	02-707-419
RNase Away	Molecular BioProducts	7005-11
T4 Polynucleotide Kinase	New England BioLabs	M0201S
T4 Polynucleotide Kinase Reaction Buffer	New England BioLabs	B0201S	This is solution A
Triflouroacetic Acid	Alfa Aesar	76-05-1
Xrn-1 exoribonuclease	Expressed in house	See ref. 20
ZipTip Pipette Tips for Sample preparation	Millipore	ZTC 18S 096	10 µL pipette tips loaded with a C18 standard 0.6 µL bed