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Quantum Chemical Calculations and Experimental Validation of the Photoclick Reaction for Fluorescent Labeling of the 5' cap of Eukaryotic mRNAs.

Stummer D, Herrmann C, Rentmeister A - ChemistryOpen (2015)

Bottom Line: To elucidate whether the resulting N (2)-modified 5' cap is a suitable dipolarophile for photoclick reactions, we used Kohn-Sham density functional theory (KS-DFT) and calculated the HOMO and LUMO energies of this molecule and nitrile imines.Our in silico studies suggested that combining enzymatic allylation of the cap with subsequent labeling in a photoclick reaction was feasible.Our approach generates a turn-on fluorophore site-specifically at the 5' cap and therefore presents an important step towards labeling of eukaryotic mRNAs in a bioorthogonal manner.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Westfälische Wilhelms-Universität Münster Wilhelm-Klemm-Straße 2, 48149, Münster, Germany ; Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), Westfälische Wilhelms-Universität Münster Wilhelm-Klemm-Straße 2, 48149, Münster, Germany.

ABSTRACT
Bioorthogonal click reactions are powerful tools to specifically label biomolecules in living cells. Considerable progress has been made in site-specific labeling of proteins and glycans in complex biological systems, but equivalent methods for mRNAs are rare. We present a chemo-enzymatic approach to label the 5' cap of eukaryotic mRNAs using a bioorthogonal photoclick reaction. Herein, the N7-methylated guanosine of the 5' cap is enzymatically equipped with an allyl group using a variant of the trimethylguanosine synthase 2 from Giardia lamblia (GlaTgs2). To elucidate whether the resulting N (2)-modified 5' cap is a suitable dipolarophile for photoclick reactions, we used Kohn-Sham density functional theory (KS-DFT) and calculated the HOMO and LUMO energies of this molecule and nitrile imines. Our in silico studies suggested that combining enzymatic allylation of the cap with subsequent labeling in a photoclick reaction was feasible. This could be experimentally validated. Our approach generates a turn-on fluorophore site-specifically at the 5' cap and therefore presents an important step towards labeling of eukaryotic mRNAs in a bioorthogonal manner.

No MeSH data available.


Possible modifications of the mRNA cap catalyzed by the GlaTgs2 variant. The GlaTgs2 variant can transfer several click reactive handles to the mRNA cap (R1=pppA) making it amenable to CuAAC, SPAAC, and thiol-ene click reactions. The yield of enzymatic conversions decreases with increasing size of the transferred moiety (X).
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sch01: Possible modifications of the mRNA cap catalyzed by the GlaTgs2 variant. The GlaTgs2 variant can transfer several click reactive handles to the mRNA cap (R1=pppA) making it amenable to CuAAC, SPAAC, and thiol-ene click reactions. The yield of enzymatic conversions decreases with increasing size of the transferred moiety (X).

Mentions: We previously described a variant of trimethylguanosine synthase 2 from Giardia lamblia (GlaTgs2) with improved activity on analogs of the cosubstrate S-adenosyl-l-methionine that can be used to transfer alkene, alkyne, or azido groups to the 5’ cap of RNA.32,33 We could show that enzymatic introduction of alkene, alkyne, or azido moieties at N2 gives access to further derivatization of the cap in CuAAC, thiol-ene, or SPAAC reactions.30,31 The yield of the enzymatic step currently depends on the cosubstrate and decreases with larger substituents (Scheme 1).


Quantum Chemical Calculations and Experimental Validation of the Photoclick Reaction for Fluorescent Labeling of the 5' cap of Eukaryotic mRNAs.

Stummer D, Herrmann C, Rentmeister A - ChemistryOpen (2015)

Possible modifications of the mRNA cap catalyzed by the GlaTgs2 variant. The GlaTgs2 variant can transfer several click reactive handles to the mRNA cap (R1=pppA) making it amenable to CuAAC, SPAAC, and thiol-ene click reactions. The yield of enzymatic conversions decreases with increasing size of the transferred moiety (X).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4522179&req=5

sch01: Possible modifications of the mRNA cap catalyzed by the GlaTgs2 variant. The GlaTgs2 variant can transfer several click reactive handles to the mRNA cap (R1=pppA) making it amenable to CuAAC, SPAAC, and thiol-ene click reactions. The yield of enzymatic conversions decreases with increasing size of the transferred moiety (X).
Mentions: We previously described a variant of trimethylguanosine synthase 2 from Giardia lamblia (GlaTgs2) with improved activity on analogs of the cosubstrate S-adenosyl-l-methionine that can be used to transfer alkene, alkyne, or azido groups to the 5’ cap of RNA.32,33 We could show that enzymatic introduction of alkene, alkyne, or azido moieties at N2 gives access to further derivatization of the cap in CuAAC, thiol-ene, or SPAAC reactions.30,31 The yield of the enzymatic step currently depends on the cosubstrate and decreases with larger substituents (Scheme 1).

Bottom Line: To elucidate whether the resulting N (2)-modified 5' cap is a suitable dipolarophile for photoclick reactions, we used Kohn-Sham density functional theory (KS-DFT) and calculated the HOMO and LUMO energies of this molecule and nitrile imines.Our in silico studies suggested that combining enzymatic allylation of the cap with subsequent labeling in a photoclick reaction was feasible.Our approach generates a turn-on fluorophore site-specifically at the 5' cap and therefore presents an important step towards labeling of eukaryotic mRNAs in a bioorthogonal manner.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Westfälische Wilhelms-Universität Münster Wilhelm-Klemm-Straße 2, 48149, Münster, Germany ; Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), Westfälische Wilhelms-Universität Münster Wilhelm-Klemm-Straße 2, 48149, Münster, Germany.

ABSTRACT
Bioorthogonal click reactions are powerful tools to specifically label biomolecules in living cells. Considerable progress has been made in site-specific labeling of proteins and glycans in complex biological systems, but equivalent methods for mRNAs are rare. We present a chemo-enzymatic approach to label the 5' cap of eukaryotic mRNAs using a bioorthogonal photoclick reaction. Herein, the N7-methylated guanosine of the 5' cap is enzymatically equipped with an allyl group using a variant of the trimethylguanosine synthase 2 from Giardia lamblia (GlaTgs2). To elucidate whether the resulting N (2)-modified 5' cap is a suitable dipolarophile for photoclick reactions, we used Kohn-Sham density functional theory (KS-DFT) and calculated the HOMO and LUMO energies of this molecule and nitrile imines. Our in silico studies suggested that combining enzymatic allylation of the cap with subsequent labeling in a photoclick reaction was feasible. This could be experimentally validated. Our approach generates a turn-on fluorophore site-specifically at the 5' cap and therefore presents an important step towards labeling of eukaryotic mRNAs in a bioorthogonal manner.

No MeSH data available.