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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.
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.
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 to over three decades8, where several limitations were noted. This technique has now evolved to a reliable, commonly used means to characterize both biopolymers9 and identify/understand chemical and biochemical reactions, e.g., characterization of platinated sites in RNA10, identification of RNA fragments following strand cleavage11,12, or formation of protein-DNA cross-links13. Thus, it is valuable to illustrate and highlight important aspects of using this technique. The basics of MALDI-TOF have been described in video format as well14 and will not be further elaborated herein. Furthermore, its application in a DNA or protein context has been previously described and illustrated in the said format15,16,17.
The protocol for detecting RNA fragments formed after enzymatic hydrolysis is reported herein. The experimental model was chosen based on a recent finding published by our group18, where MALDI-TOF was used to determine the unique reactivity between the exoribonuclease Xrn-1 and oligonucleotides of RNA containing the oxidative lesion 8-oxoG. The 20-nucleotide long strands were obtained via solid-phase synthesis19, [5'-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5'-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU], while Xrn-1 was expressed and purified following the previously described report20. In brief, Xrn-121 is a 5'-3' exoribonuclease with various key biological roles that degrade multiple types of RNA, including oxidized RNA22. It was found that the processivity of the enzyme stalls upon encountering 8-oxoG, which led to RNA fragments containing 5'-phosphorylated ends [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]18.
Finally, it is important to note that mass spectrometry is a powerful method that, through various methodologies, can be adapted to other purposes23,24; thus, choosing the right ionization method as well as other experimental set up is of utmost importance.
RNase-free ultra pure water (Table 1) was used for the present study.
1. Concentration determination of RNA solution
2. Hydrolysis of oligonucleotides of RNA by Xrn-1
3. Desalting of RNA solution, MALDI-TOF plate spotting
4. Data acquisition and processing
NOTE: In general, THAP requires higher laser power to achieve ionization. Additionally, oligos can be difficult to ionize without fragmenting. Thus, it is usually necessary to use as low laser power as possible and raise detector gains and/or lower laser frequency to maximize detection and minimize fragmentation.
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, ...
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 ...
The authors have nothing to disclose.
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 Award (CU Denver) and Eureca grants (CU Denver) for support. E. G. C. acknowledges support from NIGMS, via R00GM115757. MJER acknowledges support from NIGMS, via 1R15GM132816. K. B. acknowledges resource ID: SCR_021758. The work was also supported by a Teacher-Scholar Award (MJER), TH-21-028, from the Henry Dreyfus Foundation.
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 |
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