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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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-TOF1,2,3 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 .......

Representative 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 .......

Acknowledgements

It is important to note that this work was a collaborative effort between three institutions, two research groups, and one core facility. The distribution and workload were carried out as follows: Protein (Xrn-1) expression was carried out at the University of Denver (Denver, CO). Oligonucleotide synthesis, quantification, and experimentation (mainly enzymatic degradation) were conducted at the University of Colorado Denver (Denver, CO). Optimization was also carried out there. MALDI-TOF spotting, acquisition, and analysis were carried out at the Analytical Resources Core Facility at Colorado State University. (Fort Collins, CO). SS would like to acknowledge a UROP Aw....

Materials

NameCompanyCatalog NumberComments
0.6 mL MCT Graduated VioletFisher Scientific05-408-127
6’-Trihydroxyacetophenone monohydrate 98%Sigma Aldrich480-66-0
Acetonitrile 99.9%, HPLC gradeFisher Scientific75-05-8
Adenosine triphosphate, 10 mMNew Englang BioscienceP0756S
Ammonium citrate, dibasic 98%Sigma Aldrich3012-65-5
Ammonium Fluoride 98.0%, ACS gradeAlfa Aesar12125-01-8
Bruker bacterial test standardBruker Daltonics8255343
Commercial source of Xrn-1New England BioLabsM0338S
Diethyl pyrocarbonate, 97%ACROS OrganicsA0368487
Flex analysis softwareBruker daltonicsFlexAnalysis software version 3.4, Bruker Daltonics
Lambda 365 UV-vis spectrophotometerPerkin Elmer
MALDI plate: MSP 96 ground steel targetBruker Daltonics280799
Mass SpectrometerBrukerMicroflex LRFTOF mass spectrometer (Bruker Daltonics, Billerica, MA)
Mili-Q IQ 7000Milipore SigmaA 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 CorporationOD010C33filter media, Omega (modified polyethersulfone) 10 K pore size
NEBuffer 3New England BiolabsB7003SThis is solution B
Oligo Analyzer toolIDT-DNAhttps://www.idtdna.com/calc/analyzer
Pipette tips P10Fisher Scientific02-707-441
Pipette tips P200Fisher Scientific02-707-419
RNase AwayMolecular BioProducts7005-11
T4 Polynucleotide KinaseNew England BioLabsM0201S
T4 Polynucleotide Kinase Reaction BufferNew England BioLabsB0201SThis is solution A
Triflouroacetic AcidAlfa Aesar76-05-1
Xrn-1 exoribonucleaseExpressed in houseSee ref. 20
ZipTip Pipette Tips for Sample preparationMilliporeZTC 18S 09610 µL pipette tips loaded with a C18 standard 0.6 µL bed

References

  1. Tanaka, K., et al. Protein and polymer analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry. 2 (8), 151-153 (1988).
  2. Karas, M., Hillenkamp, F.

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