Rapid Determination of RNA Modifications in Consensus Motifs by Nuclease Protection with Ion-Tagged Oligonucleotide Probes and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry
The reversible and substoichiometric modification of RNA has recently emerged as an additional layer of translational regulation in normal biological function and disease. Modifications are often enzymatically deposited in and removed from short (~5 nt) consensus motif sequences to carefully control...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-06-01
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| Series: | Genes |
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| Online Access: | https://www.mdpi.com/2073-4425/13/6/1008 |
| _version_ | 1797487117357547520 |
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| author | Madeline E. Melzer Jonathan V. Sweedler Kevin D. Clark |
| author_facet | Madeline E. Melzer Jonathan V. Sweedler Kevin D. Clark |
| author_sort | Madeline E. Melzer |
| collection | DOAJ |
| description | The reversible and substoichiometric modification of RNA has recently emerged as an additional layer of translational regulation in normal biological function and disease. Modifications are often enzymatically deposited in and removed from short (~5 nt) consensus motif sequences to carefully control the translational output of the cell. Although characterization of modification occupancy at consensus motifs can be accomplished using RNA sequencing methods, these approaches are generally time-consuming and do not directly detect post-transcriptional modifications. Here, we present a nuclease protection assay coupled with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to rapidly characterize modifications in consensus motifs, such as GGACU, which frequently harbor N6-methyladenosine (m<sup>6</sup>A). While conventional nuclease protection methods rely on long (~30 nt) oligonucleotide probes that preclude the global assessment of consensus motif modification stoichiometry, we investigated a series of ion-tagged oligonucleotide (ITO) probes and found that a benzylimidazolium-functionalized ITO (ABzIM-ITO) conferred significantly improved nuclease resistance for GGACU targets. After optimizing the conditions of the nuclease protection assay, we applied the ITO and MALDI-MS-based method for determining the stoichiometry of GG(m<sup>6</sup>A)CU and GGACU in RNA mixtures. Overall, the ITO-based nuclease protection and MALDI-MS method constitutes a rapid and promising approach for determining modification stoichiometries of consensus motifs. |
| first_indexed | 2024-03-09T23:43:41Z |
| format | Article |
| id | doaj.art-47fc18f6cac443d5acfcec94c249d23b |
| institution | Directory Open Access Journal |
| issn | 2073-4425 |
| language | English |
| last_indexed | 2024-03-09T23:43:41Z |
| publishDate | 2022-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Genes |
| spelling | doaj.art-47fc18f6cac443d5acfcec94c249d23b2023-11-23T16:47:46ZengMDPI AGGenes2073-44252022-06-01136100810.3390/genes13061008Rapid Determination of RNA Modifications in Consensus Motifs by Nuclease Protection with Ion-Tagged Oligonucleotide Probes and Matrix-Assisted Laser Desorption Ionization Mass SpectrometryMadeline E. Melzer0Jonathan V. Sweedler1Kevin D. Clark2Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USADepartment of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USABeckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USAThe reversible and substoichiometric modification of RNA has recently emerged as an additional layer of translational regulation in normal biological function and disease. Modifications are often enzymatically deposited in and removed from short (~5 nt) consensus motif sequences to carefully control the translational output of the cell. Although characterization of modification occupancy at consensus motifs can be accomplished using RNA sequencing methods, these approaches are generally time-consuming and do not directly detect post-transcriptional modifications. Here, we present a nuclease protection assay coupled with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to rapidly characterize modifications in consensus motifs, such as GGACU, which frequently harbor N6-methyladenosine (m<sup>6</sup>A). While conventional nuclease protection methods rely on long (~30 nt) oligonucleotide probes that preclude the global assessment of consensus motif modification stoichiometry, we investigated a series of ion-tagged oligonucleotide (ITO) probes and found that a benzylimidazolium-functionalized ITO (ABzIM-ITO) conferred significantly improved nuclease resistance for GGACU targets. After optimizing the conditions of the nuclease protection assay, we applied the ITO and MALDI-MS-based method for determining the stoichiometry of GG(m<sup>6</sup>A)CU and GGACU in RNA mixtures. Overall, the ITO-based nuclease protection and MALDI-MS method constitutes a rapid and promising approach for determining modification stoichiometries of consensus motifs.https://www.mdpi.com/2073-4425/13/6/1008RNA modificationsmatrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)m<sup>6</sup>A consensus motifion-tagged oligonucleotides (ITOs)RNA modification stoichiometrynuclease protection |
| spellingShingle | Madeline E. Melzer Jonathan V. Sweedler Kevin D. Clark Rapid Determination of RNA Modifications in Consensus Motifs by Nuclease Protection with Ion-Tagged Oligonucleotide Probes and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Genes RNA modifications matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) m<sup>6</sup>A consensus motif ion-tagged oligonucleotides (ITOs) RNA modification stoichiometry nuclease protection |
| title | Rapid Determination of RNA Modifications in Consensus Motifs by Nuclease Protection with Ion-Tagged Oligonucleotide Probes and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry |
| title_full | Rapid Determination of RNA Modifications in Consensus Motifs by Nuclease Protection with Ion-Tagged Oligonucleotide Probes and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry |
| title_fullStr | Rapid Determination of RNA Modifications in Consensus Motifs by Nuclease Protection with Ion-Tagged Oligonucleotide Probes and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry |
| title_full_unstemmed | Rapid Determination of RNA Modifications in Consensus Motifs by Nuclease Protection with Ion-Tagged Oligonucleotide Probes and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry |
| title_short | Rapid Determination of RNA Modifications in Consensus Motifs by Nuclease Protection with Ion-Tagged Oligonucleotide Probes and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry |
| title_sort | rapid determination of rna modifications in consensus motifs by nuclease protection with ion tagged oligonucleotide probes and matrix assisted laser desorption ionization mass spectrometry |
| topic | RNA modifications matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) m<sup>6</sup>A consensus motif ion-tagged oligonucleotides (ITOs) RNA modification stoichiometry nuclease protection |
| url | https://www.mdpi.com/2073-4425/13/6/1008 |
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