Chemoenzymatic labeling of RNA to enrich, detect and identify methyltransferase-target sites
Copyright © 2021 Elsevier Inc. All rights reserved..
The RNA methyltransferase (MTase) complex METTL3-METTL14 transfers methyl groups from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenosines within its consensus sequence, the DRACH motif (D=A, G, U; R=A, G; H=A, C, U). Interestingly, this MTase complex shows remarkable promiscuity regarding the cosubstrate. This can be exploited to install nonnatural modifications, like clickable or photocaging groups. Clickable groups are widely used for subsequent functionalization and open a broad range of possibilities for downstream applications. Here, we elaborate on click chemistry for coupling of RNA to biotin to enrich MTase targets via streptavidin-coated magnetic beads. Importantly, after clicking and coupling to beads the modification becomes sterically demanding and stalls reverse transcriptases, leading to termination adjacent to the MTase target site. Using radioactively labeled primers in the reverse transcription, the modified position can be precisely identified on a sequencing gel via phosphor imaging.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:658 |
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| Enthalten in: |
Methods in enzymology - 658(2021) vom: 14., Seite 161-190 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Kueck, Nadine A [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 22.09.2021 Date Revised 22.09.2021 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/bs.mie.2021.06.006 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Copyright © 2021 Elsevier Inc. All rights reserved. | ||
| 520 | |a The RNA methyltransferase (MTase) complex METTL3-METTL14 transfers methyl groups from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenosines within its consensus sequence, the DRACH motif (D=A, G, U; R=A, G; H=A, C, U). Interestingly, this MTase complex shows remarkable promiscuity regarding the cosubstrate. This can be exploited to install nonnatural modifications, like clickable or photocaging groups. Clickable groups are widely used for subsequent functionalization and open a broad range of possibilities for downstream applications. Here, we elaborate on click chemistry for coupling of RNA to biotin to enrich MTase targets via streptavidin-coated magnetic beads. Importantly, after clicking and coupling to beads the modification becomes sterically demanding and stalls reverse transcriptases, leading to termination adjacent to the MTase target site. Using radioactively labeled primers in the reverse transcription, the modified position can be precisely identified on a sequencing gel via phosphor imaging | ||
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| 700 | 1 | |a Ovcharenko, Anna |e verfasserin |4 aut | |
| 700 | 1 | |a Hartstock, Katja |e verfasserin |4 aut | |
| 700 | 1 | |a Cornelissen, Nicolas V |e verfasserin |4 aut | |
| 700 | 1 | |a Rentmeister, Andrea |e verfasserin |4 aut | |
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