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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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Determination of single nucleotide incorporation efficiencies for all 2′-N3-2′-dNTPs with three different RNA sequences under conditions optimized for 4 or 5 µM (only 2′-N3-2′-dATP) RNA. RNA1, 2, and 3 were subjected to PAP reactions under optimized conditions (in triplicate, except RNA3 + 2′-N3-2′-dGTP—duplicate). Analysis by 15% seqPAGE. All radioactive bands were quantified with ImageQuant software and the percentages of singly modified product were calculated for all samples. Of these, average values are shown. Error bars indicate 1 SD.
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gks062-F2: Determination of single nucleotide incorporation efficiencies for all 2′-N3-2′-dNTPs with three different RNA sequences under conditions optimized for 4 or 5 µM (only 2′-N3-2′-dATP) RNA. RNA1, 2, and 3 were subjected to PAP reactions under optimized conditions (in triplicate, except RNA3 + 2′-N3-2′-dGTP—duplicate). Analysis by 15% seqPAGE. All radioactive bands were quantified with ImageQuant software and the percentages of singly modified product were calculated for all samples. Of these, average values are shown. Error bars indicate 1 SD.

Mentions: Figure 2 shows a triplicate determination of single nucleotide incorporation efficiencies under the optimized conditions (Table 2) for each of the three test sequences. In 11 out of the 12 combinations, single-addition product yields ≥75% were achieved. Altogether, we were able to identify conditions under which single nucleotide incorporation is prevalent for each of the 2′-modified NTPs, requiring relatively short reaction times and preserving the integrity of the RNA sequences.Figure 2.


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)

Determination of single nucleotide incorporation efficiencies for all 2′-N3-2′-dNTPs with three different RNA sequences under conditions optimized for 4 or 5 µM (only 2′-N3-2′-dATP) RNA. RNA1, 2, and 3 were subjected to PAP reactions under optimized conditions (in triplicate, except RNA3 + 2′-N3-2′-dGTP—duplicate). Analysis by 15% seqPAGE. All radioactive bands were quantified with ImageQuant software and the percentages of singly modified product were calculated for all samples. Of these, average values are shown. Error bars indicate 1 SD.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks062-F2: Determination of single nucleotide incorporation efficiencies for all 2′-N3-2′-dNTPs with three different RNA sequences under conditions optimized for 4 or 5 µM (only 2′-N3-2′-dATP) RNA. RNA1, 2, and 3 were subjected to PAP reactions under optimized conditions (in triplicate, except RNA3 + 2′-N3-2′-dGTP—duplicate). Analysis by 15% seqPAGE. All radioactive bands were quantified with ImageQuant software and the percentages of singly modified product were calculated for all samples. Of these, average values are shown. Error bars indicate 1 SD.
Mentions: Figure 2 shows a triplicate determination of single nucleotide incorporation efficiencies under the optimized conditions (Table 2) for each of the three test sequences. In 11 out of the 12 combinations, single-addition product yields ≥75% were achieved. Altogether, we were able to identify conditions under which single nucleotide incorporation is prevalent for each of the 2′-modified NTPs, requiring relatively short reaction times and preserving the integrity of the RNA sequences.Figure 2.

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