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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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Related in: MedlinePlus

Fluorescent labeling of a natural RNA sample. A total RNA isolate from E. coli was reacted with 2′-N3-2′-dUTP and yeast PAP, and further subjected to CuAAC with Alexa Fluor 647 alkyne. Analysis by 8% denaturing PAGE. SYBR Gold scan (left panel), Alexa Fluor 647 scan (middle panel) and overlay (green: radioactivity; magenta: fluorescence; white: both; right panel) are given. The two main bands represent 23S and 16S ribosomal RNA.
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gks062-F5: Fluorescent labeling of a natural RNA sample. A total RNA isolate from E. coli was reacted with 2′-N3-2′-dUTP and yeast PAP, and further subjected to CuAAC with Alexa Fluor 647 alkyne. Analysis by 8% denaturing PAGE. SYBR Gold scan (left panel), Alexa Fluor 647 scan (middle panel) and overlay (green: radioactivity; magenta: fluorescence; white: both; right panel) are given. The two main bands represent 23S and 16S ribosomal RNA.

Mentions: To test the robustness of our labeling approach and its applicability to RNA from biological sources, we isolated total RNA from E. coli, reacted this isolate with 2′-N3-2′-dUTP and yeast PAP. We employed a 30-min incubation time to assure that every sequence carries at least one modified nucleotide, and performed the CuAAC with Alexa Fluor 647 alkyne afterwards. To prevent degradation of the total RNA sample, both, reaction time and temperature were decreased (30 min incubation at 25°C). In the PAGE analysis (Figure 5), the appearance of fluorescent bands that perfectly overlay with the bands visible after non-specific RNA staining and the absence of degradation products indicate the applicability of this labeling approach toward long natural RNA.Figure 5.


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)

Fluorescent labeling of a natural RNA sample. A total RNA isolate from E. coli was reacted with 2′-N3-2′-dUTP and yeast PAP, and further subjected to CuAAC with Alexa Fluor 647 alkyne. Analysis by 8% denaturing PAGE. SYBR Gold scan (left panel), Alexa Fluor 647 scan (middle panel) and overlay (green: radioactivity; magenta: fluorescence; white: both; right panel) are given. The two main bands represent 23S and 16S ribosomal RNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks062-F5: Fluorescent labeling of a natural RNA sample. A total RNA isolate from E. coli was reacted with 2′-N3-2′-dUTP and yeast PAP, and further subjected to CuAAC with Alexa Fluor 647 alkyne. Analysis by 8% denaturing PAGE. SYBR Gold scan (left panel), Alexa Fluor 647 scan (middle panel) and overlay (green: radioactivity; magenta: fluorescence; white: both; right panel) are given. The two main bands represent 23S and 16S ribosomal RNA.
Mentions: To test the robustness of our labeling approach and its applicability to RNA from biological sources, we isolated total RNA from E. coli, reacted this isolate with 2′-N3-2′-dUTP and yeast PAP. We employed a 30-min incubation time to assure that every sequence carries at least one modified nucleotide, and performed the CuAAC with Alexa Fluor 647 alkyne afterwards. To prevent degradation of the total RNA sample, both, reaction time and temperature were decreased (30 min incubation at 25°C). In the PAGE analysis (Figure 5), the appearance of fluorescent bands that perfectly overlay with the bands visible after non-specific RNA staining and the absence of degradation products indicate the applicability of this labeling approach toward long natural RNA.Figure 5.

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