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Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling.

McDonald RI, Guilinger JP, Mukherji S, Curtis EA, Lee WI, Liu DR - Nat. Chem. Biol. (2014)

Bottom Line: We developed this catalytic RNA into a general tool to selectively conjugate a small molecule to an RNA of interest.This strategy enabled up to 500-fold enrichment of target RNA from total mammalian RNA or from cell lysate.We demonstrated the utility of this approach by selectively capturing proteins in yeast cell lysate that bind the ASH1 mRNA.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
Probes that form covalent bonds with RNA molecules on the basis of their chemical reactivity would advance our ability to study the transcriptome. We developed a set of electrophilic activity-based RNA probes designed to react with unusually nucleophilic RNAs. We used these probes to identify reactive genome-encoded RNAs, resulting in the discovery of a 42-nt catalytic RNA from an archaebacterium that reacts with a 2,3-disubstituted epoxide at N7 of a specific guanosine. Detailed characterization of the catalytic RNA revealed the structural requirements for reactivity. We developed this catalytic RNA into a general tool to selectively conjugate a small molecule to an RNA of interest. This strategy enabled up to 500-fold enrichment of target RNA from total mammalian RNA or from cell lysate. We demonstrated the utility of this approach by selectively capturing proteins in yeast cell lysate that bind the ASH1 mRNA.

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Application of the epoxide-opening catalytic RNA to enrich RNAs of interest from total cellular RNA and to capture RNA-binding proteins(a) Transcriptional fusion of a self-labeling catalytic RNA to an RNA of interest may enable selective, covalent RNA modification in a complex biological sample. (b) Total RNA from HEK 293T cells was reacted with epoxide-azide 14, followed by DBCO-TAMRA. Total RNA was analyzed by PAGE and TAMRA-modified RNAs were visualized by fluorescence imaging. Lanes 1 and 6: in vitro transcribed catalytic RNA-fused 5S rRNA containing one or three copies of the catalytic RNA, respectively, rather than cellular RNA. Lanes 2 and 3: the inactive C9-G35 mutant RNA. Lanes 4–8: 5S rRNA fused to one copy (lanes 4–5) or three copies (lanes 6–8) of the active optimized catalytic RNA. Bands at the top of the gel result from incomplete removal of excess DBCO-TAMRA probe or background labeling of cellular rRNAs/mRNAs. The complete gel is shown in Supplementary Figure 14. (c) Western blot probing the presence of three known ASH1 mRNA-binding proteins (Puf6, Khd1, and She2) and one non-binding protein control (Guk1) in yeast cell lysate. Lanes 1 and 2: Lysate incubated overnight with streptavidin-coated magnetic beads only (lane 1) or pre-incubated with 5 µg of epoxide 1-modified ASH1-catalytic RNA (lane 2). Unbound proteins were washed away and captured proteins were eluted at 95 °C. Lane 3: Input lysate prior to incubation with beads. The complete gel is shown in Supplementary Figure 15.
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Figure 4: Application of the epoxide-opening catalytic RNA to enrich RNAs of interest from total cellular RNA and to capture RNA-binding proteins(a) Transcriptional fusion of a self-labeling catalytic RNA to an RNA of interest may enable selective, covalent RNA modification in a complex biological sample. (b) Total RNA from HEK 293T cells was reacted with epoxide-azide 14, followed by DBCO-TAMRA. Total RNA was analyzed by PAGE and TAMRA-modified RNAs were visualized by fluorescence imaging. Lanes 1 and 6: in vitro transcribed catalytic RNA-fused 5S rRNA containing one or three copies of the catalytic RNA, respectively, rather than cellular RNA. Lanes 2 and 3: the inactive C9-G35 mutant RNA. Lanes 4–8: 5S rRNA fused to one copy (lanes 4–5) or three copies (lanes 6–8) of the active optimized catalytic RNA. Bands at the top of the gel result from incomplete removal of excess DBCO-TAMRA probe or background labeling of cellular rRNAs/mRNAs. The complete gel is shown in Supplementary Figure 14. (c) Western blot probing the presence of three known ASH1 mRNA-binding proteins (Puf6, Khd1, and She2) and one non-binding protein control (Guk1) in yeast cell lysate. Lanes 1 and 2: Lysate incubated overnight with streptavidin-coated magnetic beads only (lane 1) or pre-incubated with 5 µg of epoxide 1-modified ASH1-catalytic RNA (lane 2). Unbound proteins were washed away and captured proteins were eluted at 95 °C. Lane 3: Input lysate prior to incubation with beads. The complete gel is shown in Supplementary Figure 15.

Mentions: To establish the ability of the catalytic RNA to enrich an RNA of interest from total cellular RNA (Fig. 4a), we transfected the 5S rRNA–catalytic RNA construct into HEK 293T cells and isolated total RNA. As a control we used a construct in which the reactive G9 and base C35, predicted to pair with G9, were swapped to yield an inactive isomeric mutant catalytic RNA predicted to adopt the same secondary structure. Total cellular RNA was incubated for 5 hours with azide epoxide 14, followed by copper-free click chemistry using dibenzocyclooctyne–TAMRA (DBCO–TAMRA) to install a TAMRA fluorophore and PAGE analysis (Fig. 4b). While no TAMRA-bound 5S rRNA product was generated from the mutant catalytic RNA, we observed a strong fluorescent band of the expected size for the sample containing the active catalytic RNA (Fig. 4b). Repeating this experiment with a construct containing three consecutive copies of the catalytic RNA (5S rRNA–catalytic RNA3) resulted in 3.2-fold higher fluorescence signal (Fig. 4b).


Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling.

McDonald RI, Guilinger JP, Mukherji S, Curtis EA, Lee WI, Liu DR - Nat. Chem. Biol. (2014)

Application of the epoxide-opening catalytic RNA to enrich RNAs of interest from total cellular RNA and to capture RNA-binding proteins(a) Transcriptional fusion of a self-labeling catalytic RNA to an RNA of interest may enable selective, covalent RNA modification in a complex biological sample. (b) Total RNA from HEK 293T cells was reacted with epoxide-azide 14, followed by DBCO-TAMRA. Total RNA was analyzed by PAGE and TAMRA-modified RNAs were visualized by fluorescence imaging. Lanes 1 and 6: in vitro transcribed catalytic RNA-fused 5S rRNA containing one or three copies of the catalytic RNA, respectively, rather than cellular RNA. Lanes 2 and 3: the inactive C9-G35 mutant RNA. Lanes 4–8: 5S rRNA fused to one copy (lanes 4–5) or three copies (lanes 6–8) of the active optimized catalytic RNA. Bands at the top of the gel result from incomplete removal of excess DBCO-TAMRA probe or background labeling of cellular rRNAs/mRNAs. The complete gel is shown in Supplementary Figure 14. (c) Western blot probing the presence of three known ASH1 mRNA-binding proteins (Puf6, Khd1, and She2) and one non-binding protein control (Guk1) in yeast cell lysate. Lanes 1 and 2: Lysate incubated overnight with streptavidin-coated magnetic beads only (lane 1) or pre-incubated with 5 µg of epoxide 1-modified ASH1-catalytic RNA (lane 2). Unbound proteins were washed away and captured proteins were eluted at 95 °C. Lane 3: Input lysate prior to incubation with beads. The complete gel is shown in Supplementary Figure 15.
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Figure 4: Application of the epoxide-opening catalytic RNA to enrich RNAs of interest from total cellular RNA and to capture RNA-binding proteins(a) Transcriptional fusion of a self-labeling catalytic RNA to an RNA of interest may enable selective, covalent RNA modification in a complex biological sample. (b) Total RNA from HEK 293T cells was reacted with epoxide-azide 14, followed by DBCO-TAMRA. Total RNA was analyzed by PAGE and TAMRA-modified RNAs were visualized by fluorescence imaging. Lanes 1 and 6: in vitro transcribed catalytic RNA-fused 5S rRNA containing one or three copies of the catalytic RNA, respectively, rather than cellular RNA. Lanes 2 and 3: the inactive C9-G35 mutant RNA. Lanes 4–8: 5S rRNA fused to one copy (lanes 4–5) or three copies (lanes 6–8) of the active optimized catalytic RNA. Bands at the top of the gel result from incomplete removal of excess DBCO-TAMRA probe or background labeling of cellular rRNAs/mRNAs. The complete gel is shown in Supplementary Figure 14. (c) Western blot probing the presence of three known ASH1 mRNA-binding proteins (Puf6, Khd1, and She2) and one non-binding protein control (Guk1) in yeast cell lysate. Lanes 1 and 2: Lysate incubated overnight with streptavidin-coated magnetic beads only (lane 1) or pre-incubated with 5 µg of epoxide 1-modified ASH1-catalytic RNA (lane 2). Unbound proteins were washed away and captured proteins were eluted at 95 °C. Lane 3: Input lysate prior to incubation with beads. The complete gel is shown in Supplementary Figure 15.
Mentions: To establish the ability of the catalytic RNA to enrich an RNA of interest from total cellular RNA (Fig. 4a), we transfected the 5S rRNA–catalytic RNA construct into HEK 293T cells and isolated total RNA. As a control we used a construct in which the reactive G9 and base C35, predicted to pair with G9, were swapped to yield an inactive isomeric mutant catalytic RNA predicted to adopt the same secondary structure. Total cellular RNA was incubated for 5 hours with azide epoxide 14, followed by copper-free click chemistry using dibenzocyclooctyne–TAMRA (DBCO–TAMRA) to install a TAMRA fluorophore and PAGE analysis (Fig. 4b). While no TAMRA-bound 5S rRNA product was generated from the mutant catalytic RNA, we observed a strong fluorescent band of the expected size for the sample containing the active catalytic RNA (Fig. 4b). Repeating this experiment with a construct containing three consecutive copies of the catalytic RNA (5S rRNA–catalytic RNA3) resulted in 3.2-fold higher fluorescence signal (Fig. 4b).

Bottom Line: We developed this catalytic RNA into a general tool to selectively conjugate a small molecule to an RNA of interest.This strategy enabled up to 500-fold enrichment of target RNA from total mammalian RNA or from cell lysate.We demonstrated the utility of this approach by selectively capturing proteins in yeast cell lysate that bind the ASH1 mRNA.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
Probes that form covalent bonds with RNA molecules on the basis of their chemical reactivity would advance our ability to study the transcriptome. We developed a set of electrophilic activity-based RNA probes designed to react with unusually nucleophilic RNAs. We used these probes to identify reactive genome-encoded RNAs, resulting in the discovery of a 42-nt catalytic RNA from an archaebacterium that reacts with a 2,3-disubstituted epoxide at N7 of a specific guanosine. Detailed characterization of the catalytic RNA revealed the structural requirements for reactivity. We developed this catalytic RNA into a general tool to selectively conjugate a small molecule to an RNA of interest. This strategy enabled up to 500-fold enrichment of target RNA from total mammalian RNA or from cell lysate. We demonstrated the utility of this approach by selectively capturing proteins in yeast cell lysate that bind the ASH1 mRNA.

Show MeSH