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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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Incorporation of different N3-modified NTPs by different PAPs. (A) Overview of the modified NTPs employed in this study. (B) Schematic representation of the addition of a 2′-N3-modified nucleotide to the 3′-terminus of an RNA sequence by PAP. (C) Addition of various N3-modified nucleotides by yeast and E. coli PAP to the 3′-terminus of RNA1. N.R.: no reaction control. Analysis by 18% seqPAGE. Radioactive bands are shown. Bandshifts indicate the incorporation of one or more nucleotides.
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gks062-F1: Incorporation of different N3-modified NTPs by different PAPs. (A) Overview of the modified NTPs employed in this study. (B) Schematic representation of the addition of a 2′-N3-modified nucleotide to the 3′-terminus of an RNA sequence by PAP. (C) Addition of various N3-modified nucleotides by yeast and E. coli PAP to the 3′-terminus of RNA1. N.R.: no reaction control. Analysis by 18% seqPAGE. Radioactive bands are shown. Bandshifts indicate the incorporation of one or more nucleotides.

Mentions: To achieve both, base- and backbone modifications, we employed NTPs bearing the azide modification at position C-2′, C-3′ or C-8 (see Figure 1A for an overview). We determined which of the four nucleotidyl transferases (yeast and E. coli PAP, Cid 1 PUP and TdT) are best suited to efficiently transfer the different N3-modified nucleotides to the 3′-termini of RNA (shown schematically in Figure 1B for modification of RNA with 2′-modified nucleotides and PAP). The results are summarized in Table 3.Figure 1.


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)

Incorporation of different N3-modified NTPs by different PAPs. (A) Overview of the modified NTPs employed in this study. (B) Schematic representation of the addition of a 2′-N3-modified nucleotide to the 3′-terminus of an RNA sequence by PAP. (C) Addition of various N3-modified nucleotides by yeast and E. coli PAP to the 3′-terminus of RNA1. N.R.: no reaction control. Analysis by 18% seqPAGE. Radioactive bands are shown. Bandshifts indicate the incorporation of one or more nucleotides.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks062-F1: Incorporation of different N3-modified NTPs by different PAPs. (A) Overview of the modified NTPs employed in this study. (B) Schematic representation of the addition of a 2′-N3-modified nucleotide to the 3′-terminus of an RNA sequence by PAP. (C) Addition of various N3-modified nucleotides by yeast and E. coli PAP to the 3′-terminus of RNA1. N.R.: no reaction control. Analysis by 18% seqPAGE. Radioactive bands are shown. Bandshifts indicate the incorporation of one or more nucleotides.
Mentions: To achieve both, base- and backbone modifications, we employed NTPs bearing the azide modification at position C-2′, C-3′ or C-8 (see Figure 1A for an overview). We determined which of the four nucleotidyl transferases (yeast and E. coli PAP, Cid 1 PUP and TdT) are best suited to efficiently transfer the different N3-modified nucleotides to the 3′-termini of RNA (shown schematically in Figure 1B for modification of RNA with 2′-modified nucleotides and PAP). The results are summarized in Table 3.Figure 1.

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