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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 RNA by CuAAC or SPAAC. RNA1, modified with each of the four 2′-N3-2′-dNTPs under optimized conditions and further conjugated with fluorescent dyes. Analysis by 15% seqPAGE. Radioactivity scan (upper panel), fluorescence scan (middle panel) and an overlay of both (green: radioactivity; magenta: fluorescence; white: both; lower panel) are given. (A) Conjugation with Alexa Fluor 647 alkyne by CuAAC. (B) Conjugation with DIBAC Fluor 488 by SPAAC.
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gks062-F3: Fluorescent labeling of RNA by CuAAC or SPAAC. RNA1, modified with each of the four 2′-N3-2′-dNTPs under optimized conditions and further conjugated with fluorescent dyes. Analysis by 15% seqPAGE. Radioactivity scan (upper panel), fluorescence scan (middle panel) and an overlay of both (green: radioactivity; magenta: fluorescence; white: both; lower panel) are given. (A) Conjugation with Alexa Fluor 647 alkyne by CuAAC. (B) Conjugation with DIBAC Fluor 488 by SPAAC.

Mentions: To convert the azide modification to various functional tags, we employed the CuAAC using biotin-alkyne, Alexa Fluor 488 alkyne or Alexa Fluor 647 alkyne, together with a water-soluble Cu-stabilizing ligand, THPTA (50). A Cu-stabilizing ligand is necessary to protect the biomolecule, i.e. RNA from degradation by free copper ions (57) and oxidation products of ascorbate (50). RNA modified with any one of the four 2′-N3-2′-dNTPs (Figure 3A) or with 8-N3-ATP (Supplementary Figure S9) could be efficiently conjugated with various functional moieties: fluorophores (Figure 3A) or biotin (Supplementary Figure S10). Purification of the RNA from the PAP reactions by size exclusion chromatography, ethanol or isopropanol precipitation proved to be efficient enough in removing excess azido-NTPs prior to CuAAC. Under carefully optimized conditions, we achieved high yields (from >80% up to quantitative) at RNA concentrations ranging from 50 nM to 50 µM. Yields and concentrations of the respective azide-bearing RNA, alkyne, as well as Cu(II), THPTA and ascorbate, are summarized in Table 4. In all cases, it was sufficient to incubate the reactions for 2 h at 37°C, which lead to excellent reaction yields without causing significant RNA degradation.Figure 3.


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 RNA by CuAAC or SPAAC. RNA1, modified with each of the four 2′-N3-2′-dNTPs under optimized conditions and further conjugated with fluorescent dyes. Analysis by 15% seqPAGE. Radioactivity scan (upper panel), fluorescence scan (middle panel) and an overlay of both (green: radioactivity; magenta: fluorescence; white: both; lower panel) are given. (A) Conjugation with Alexa Fluor 647 alkyne by CuAAC. (B) Conjugation with DIBAC Fluor 488 by SPAAC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks062-F3: Fluorescent labeling of RNA by CuAAC or SPAAC. RNA1, modified with each of the four 2′-N3-2′-dNTPs under optimized conditions and further conjugated with fluorescent dyes. Analysis by 15% seqPAGE. Radioactivity scan (upper panel), fluorescence scan (middle panel) and an overlay of both (green: radioactivity; magenta: fluorescence; white: both; lower panel) are given. (A) Conjugation with Alexa Fluor 647 alkyne by CuAAC. (B) Conjugation with DIBAC Fluor 488 by SPAAC.
Mentions: To convert the azide modification to various functional tags, we employed the CuAAC using biotin-alkyne, Alexa Fluor 488 alkyne or Alexa Fluor 647 alkyne, together with a water-soluble Cu-stabilizing ligand, THPTA (50). A Cu-stabilizing ligand is necessary to protect the biomolecule, i.e. RNA from degradation by free copper ions (57) and oxidation products of ascorbate (50). RNA modified with any one of the four 2′-N3-2′-dNTPs (Figure 3A) or with 8-N3-ATP (Supplementary Figure S9) could be efficiently conjugated with various functional moieties: fluorophores (Figure 3A) or biotin (Supplementary Figure S10). Purification of the RNA from the PAP reactions by size exclusion chromatography, ethanol or isopropanol precipitation proved to be efficient enough in removing excess azido-NTPs prior to CuAAC. Under carefully optimized conditions, we achieved high yields (from >80% up to quantitative) at RNA concentrations ranging from 50 nM to 50 µM. Yields and concentrations of the respective azide-bearing RNA, alkyne, as well as Cu(II), THPTA and ascorbate, are summarized in Table 4. In all cases, it was sufficient to incubate the reactions for 2 h at 37°C, which lead to excellent reaction yields without causing significant RNA degradation.Figure 3.

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