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Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry.

Winz ML, Samanta A, Benzinger D, Jäschke A - Nucleic Acids Res. (2012)

Bottom Line: Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2'-position can be incorporated site-specifically.This azide is subsequently reacted with a fluorophore alkyne.With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, Heidelberg 69120, Germany.

ABSTRACT
The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest--of either synthetic or biological origin--is modified at its 3'-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide-alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2'-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2'-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.

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Enzymatic manipulation of N3-modified 3′-termini. (A) Polyadenylation of RNA1 modified with each of the four 2′-N3-nucleotides by yeast PAP and E. coli PAP. RNA1, reacted with each of the four 2′-N3-NTPs under optimized conditions, and further reacted either with yeast PAP or with E. coli PAP in the presence of ATP. Analysis by 12% seqPAGE. (B) RNA1 modified with 8-N3-ATP at the indicated concentrations, and further subjected to polyadenylation with yeast PAP or adapter ligation. N.R.: no reaction control. Analysis by 15% seqPAGE. Asterisk indicates bands that appear as artifacts, due to the presence of a monophosphate at the 5′-terminus of the radiolabeled RNA, which was doped into the reaction for visualization. Lower running bands represent circularized RNA, while higher running bands represent products of RNA–RNA ligation (with or without adapter added). Both radioactive scans shown here have been scaled. A non-scaled representation is given in Supplementary Figure S13.
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gks062-F6: Enzymatic manipulation of N3-modified 3′-termini. (A) Polyadenylation of RNA1 modified with each of the four 2′-N3-nucleotides by yeast PAP and E. coli PAP. RNA1, reacted with each of the four 2′-N3-NTPs under optimized conditions, and further reacted either with yeast PAP or with E. coli PAP in the presence of ATP. Analysis by 12% seqPAGE. (B) RNA1 modified with 8-N3-ATP at the indicated concentrations, and further subjected to polyadenylation with yeast PAP or adapter ligation. N.R.: no reaction control. Analysis by 15% seqPAGE. Asterisk indicates bands that appear as artifacts, due to the presence of a monophosphate at the 5′-terminus of the radiolabeled RNA, which was doped into the reaction for visualization. Lower running bands represent circularized RNA, while higher running bands represent products of RNA–RNA ligation (with or without adapter added). Both radioactive scans shown here have been scaled. A non-scaled representation is given in Supplementary Figure S13.

Mentions: The results of polyadenylation and adapter ligation are summarized in Table 5. While yeast PAP could not efficiently polyadenylate most of the templates containing a 2′-N3-modified nucleotide at their 3′-termini, the polyadenylation reaction proceeded quantitatively when carried out with E. coli PAP (Figure 6A). For RNA containing 8-N3-A at the 3′-terminus (Figure 6B), near-quantitative polyadenylation by yeast PAP was observed for those sequences that contained a single 8-N3-A moiety. Interestingly, the polyadenylation reaction was inhibited when two 8-N3-A moieties were present.Figure 6.


Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry.

Winz ML, Samanta A, Benzinger D, Jäschke A - Nucleic Acids Res. (2012)

Enzymatic manipulation of N3-modified 3′-termini. (A) Polyadenylation of RNA1 modified with each of the four 2′-N3-nucleotides by yeast PAP and E. coli PAP. RNA1, reacted with each of the four 2′-N3-NTPs under optimized conditions, and further reacted either with yeast PAP or with E. coli PAP in the presence of ATP. Analysis by 12% seqPAGE. (B) RNA1 modified with 8-N3-ATP at the indicated concentrations, and further subjected to polyadenylation with yeast PAP or adapter ligation. N.R.: no reaction control. Analysis by 15% seqPAGE. Asterisk indicates bands that appear as artifacts, due to the presence of a monophosphate at the 5′-terminus of the radiolabeled RNA, which was doped into the reaction for visualization. Lower running bands represent circularized RNA, while higher running bands represent products of RNA–RNA ligation (with or without adapter added). Both radioactive scans shown here have been scaled. A non-scaled representation is given in Supplementary Figure S13.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3378897&req=5

gks062-F6: Enzymatic manipulation of N3-modified 3′-termini. (A) Polyadenylation of RNA1 modified with each of the four 2′-N3-nucleotides by yeast PAP and E. coli PAP. RNA1, reacted with each of the four 2′-N3-NTPs under optimized conditions, and further reacted either with yeast PAP or with E. coli PAP in the presence of ATP. Analysis by 12% seqPAGE. (B) RNA1 modified with 8-N3-ATP at the indicated concentrations, and further subjected to polyadenylation with yeast PAP or adapter ligation. N.R.: no reaction control. Analysis by 15% seqPAGE. Asterisk indicates bands that appear as artifacts, due to the presence of a monophosphate at the 5′-terminus of the radiolabeled RNA, which was doped into the reaction for visualization. Lower running bands represent circularized RNA, while higher running bands represent products of RNA–RNA ligation (with or without adapter added). Both radioactive scans shown here have been scaled. A non-scaled representation is given in Supplementary Figure S13.
Mentions: The results of polyadenylation and adapter ligation are summarized in Table 5. While yeast PAP could not efficiently polyadenylate most of the templates containing a 2′-N3-modified nucleotide at their 3′-termini, the polyadenylation reaction proceeded quantitatively when carried out with E. coli PAP (Figure 6A). For RNA containing 8-N3-A at the 3′-terminus (Figure 6B), near-quantitative polyadenylation by yeast PAP was observed for those sequences that contained a single 8-N3-A moiety. Interestingly, the polyadenylation reaction was inhibited when two 8-N3-A moieties were present.Figure 6.

Bottom Line: Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2'-position can be incorporated site-specifically.This azide is subsequently reacted with a fluorophore alkyne.With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, Heidelberg 69120, Germany.

ABSTRACT
The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest--of either synthetic or biological origin--is modified at its 3'-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide-alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2'-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2'-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.

Show MeSH
Related in: MedlinePlus