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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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Creating an internal fluorescent label. (A) General reaction scheme for creation of terminal (upper right corner) or internal modifications (lower right corner). An internal modification can be created by first adding an N3-modified nucleotide to the 3′-terminus of the RNA sequence, connecting this RNA to a second RNA sequence via splinted ligation, and subjecting the product, with an internal N3-modification to CuAAC. (B) Splinted ligation of RNA1 and RNA4, employing different ligases (RNL2 and DNL) under different reaction conditions (time and temperature). Analysis by 15% seqPAGE. (C) Addition of 2′-N3-guanosine to RNA1, followed by splinted ligation to RNA4 (using RNL2), and CuAAC with Alexa Fluor 488/647 alkyne, with or without the use of a helper DNA that forces the modified position into a 9-nt bulge loop. Analysis by 15% seqPAGE. Radioactivity scan (left panel), and an overlay of Alexa Fluor 488 scan (green, middle two lanes) and Alexa Fluor 647 scan (magenta, right two lanes) are shown. (D) Formation of 9 nt bulge loop to assist CuAAC. N.R.: no reaction control.
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gks062-F7: Creating an internal fluorescent label. (A) General reaction scheme for creation of terminal (upper right corner) or internal modifications (lower right corner). An internal modification can be created by first adding an N3-modified nucleotide to the 3′-terminus of the RNA sequence, connecting this RNA to a second RNA sequence via splinted ligation, and subjecting the product, with an internal N3-modification to CuAAC. (B) Splinted ligation of RNA1 and RNA4, employing different ligases (RNL2 and DNL) under different reaction conditions (time and temperature). Analysis by 15% seqPAGE. (C) Addition of 2′-N3-guanosine to RNA1, followed by splinted ligation to RNA4 (using RNL2), and CuAAC with Alexa Fluor 488/647 alkyne, with or without the use of a helper DNA that forces the modified position into a 9-nt bulge loop. Analysis by 15% seqPAGE. Radioactivity scan (left panel), and an overlay of Alexa Fluor 488 scan (green, middle two lanes) and Alexa Fluor 647 scan (magenta, right two lanes) are shown. (D) Formation of 9 nt bulge loop to assist CuAAC. N.R.: no reaction control.

Mentions: An RNA oligonucleotide modified with 2′-N3-2′-dGTP was subjected to splinted ligation (as shown schematically in Figure 7A), demonstrating the feasibility of the approach. Both, DNL and RNL2 accept the modified 3′-terminus as substrate (Figure 7B), with RNL2 showing better efficiencies than DNL. For both enzymes, an incubation for 1 h at 37°C produced significantly higher yields than 16 h at 16°C. With a 5-fold excess of the second ligation fragment and incubation times of 1 h at 37°C, ∼50% ligation efficiency could be achieved with DNL, while the ligation with RNL2 had an excellent efficiency of ∼ 80% of desired product. In contrast to this, as expected, ligation was not possible any longer when a bulkier modification like a fluorophore was attached by CuAAC prior to the ligation (Supplementary Figure S15). In this case, the modification at the 2′-position of the 3′-terminal nucleotide is much bulkier than just an azide and inhibits the ligase.Figure 7.


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)

Creating an internal fluorescent label. (A) General reaction scheme for creation of terminal (upper right corner) or internal modifications (lower right corner). An internal modification can be created by first adding an N3-modified nucleotide to the 3′-terminus of the RNA sequence, connecting this RNA to a second RNA sequence via splinted ligation, and subjecting the product, with an internal N3-modification to CuAAC. (B) Splinted ligation of RNA1 and RNA4, employing different ligases (RNL2 and DNL) under different reaction conditions (time and temperature). Analysis by 15% seqPAGE. (C) Addition of 2′-N3-guanosine to RNA1, followed by splinted ligation to RNA4 (using RNL2), and CuAAC with Alexa Fluor 488/647 alkyne, with or without the use of a helper DNA that forces the modified position into a 9-nt bulge loop. Analysis by 15% seqPAGE. Radioactivity scan (left panel), and an overlay of Alexa Fluor 488 scan (green, middle two lanes) and Alexa Fluor 647 scan (magenta, right two lanes) are shown. (D) Formation of 9 nt bulge loop to assist CuAAC. N.R.: no reaction control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks062-F7: Creating an internal fluorescent label. (A) General reaction scheme for creation of terminal (upper right corner) or internal modifications (lower right corner). An internal modification can be created by first adding an N3-modified nucleotide to the 3′-terminus of the RNA sequence, connecting this RNA to a second RNA sequence via splinted ligation, and subjecting the product, with an internal N3-modification to CuAAC. (B) Splinted ligation of RNA1 and RNA4, employing different ligases (RNL2 and DNL) under different reaction conditions (time and temperature). Analysis by 15% seqPAGE. (C) Addition of 2′-N3-guanosine to RNA1, followed by splinted ligation to RNA4 (using RNL2), and CuAAC with Alexa Fluor 488/647 alkyne, with or without the use of a helper DNA that forces the modified position into a 9-nt bulge loop. Analysis by 15% seqPAGE. Radioactivity scan (left panel), and an overlay of Alexa Fluor 488 scan (green, middle two lanes) and Alexa Fluor 647 scan (magenta, right two lanes) are shown. (D) Formation of 9 nt bulge loop to assist CuAAC. N.R.: no reaction control.
Mentions: An RNA oligonucleotide modified with 2′-N3-2′-dGTP was subjected to splinted ligation (as shown schematically in Figure 7A), demonstrating the feasibility of the approach. Both, DNL and RNL2 accept the modified 3′-terminus as substrate (Figure 7B), with RNL2 showing better efficiencies than DNL. For both enzymes, an incubation for 1 h at 37°C produced significantly higher yields than 16 h at 16°C. With a 5-fold excess of the second ligation fragment and incubation times of 1 h at 37°C, ∼50% ligation efficiency could be achieved with DNL, while the ligation with RNL2 had an excellent efficiency of ∼ 80% of desired product. In contrast to this, as expected, ligation was not possible any longer when a bulkier modification like a fluorophore was attached by CuAAC prior to the ligation (Supplementary Figure S15). In this case, the modification at the 2′-position of the 3′-terminal nucleotide is much bulkier than just an azide and inhibits the ligase.Figure 7.

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