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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.


Chemo-enzymatic approach for labeling the 5’ cap by the photoclick reaction. a) The capped minimal mRNA m7GpppA (1) is specifically modified by a variant of GlaTgs2, which introduces the allyl residue from 2 b at position N2, while the coproduct S-adenosylhomocysteine (SAH) is released. A reaction at 37 °C for 180 min yields N2-allyl-m7GpppA (3 b, up to 90 %). b) The product 3 b together with tetrazole 4 or 5 is irradiated at 254 nm for 5 min and left at 4 °C o/n. The 1,3-dipolar cycloaddition reaction yields fluorescent pyrazoline cycloadduct 6 or 7 (yields not determined).
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sch02: Chemo-enzymatic approach for labeling the 5’ cap by the photoclick reaction. a) The capped minimal mRNA m7GpppA (1) is specifically modified by a variant of GlaTgs2, which introduces the allyl residue from 2 b at position N2, while the coproduct S-adenosylhomocysteine (SAH) is released. A reaction at 37 °C for 180 min yields N2-allyl-m7GpppA (3 b, up to 90 %). b) The product 3 b together with tetrazole 4 or 5 is irradiated at 254 nm for 5 min and left at 4 °C o/n. The 1,3-dipolar cycloaddition reaction yields fluorescent pyrazoline cycloadduct 6 or 7 (yields not determined).

Mentions: In this work we established a chemo-enzymatic approach to fluorescently label the 5’ cap typical of eukaryotic mRNAs using a photoclick reaction (Scheme 2). The 5’ cap consists of an N7-methylated guanosine which is linked to the next nucleotide by a 5′–5′ triphosphate bridge. It is specifically recognized by trimethylguanosine synthases, which are RNA methyltransferases that transfer one or two additional methyl groups to the exocyclic N2.45–47 One such enzyme, GlaTgs2, is well characterized and transfers only a single methyl group.45,46


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)

Chemo-enzymatic approach for labeling the 5’ cap by the photoclick reaction. a) The capped minimal mRNA m7GpppA (1) is specifically modified by a variant of GlaTgs2, which introduces the allyl residue from 2 b at position N2, while the coproduct S-adenosylhomocysteine (SAH) is released. A reaction at 37 °C for 180 min yields N2-allyl-m7GpppA (3 b, up to 90 %). b) The product 3 b together with tetrazole 4 or 5 is irradiated at 254 nm for 5 min and left at 4 °C o/n. The 1,3-dipolar cycloaddition reaction yields fluorescent pyrazoline cycloadduct 6 or 7 (yields not determined).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

sch02: Chemo-enzymatic approach for labeling the 5’ cap by the photoclick reaction. a) The capped minimal mRNA m7GpppA (1) is specifically modified by a variant of GlaTgs2, which introduces the allyl residue from 2 b at position N2, while the coproduct S-adenosylhomocysteine (SAH) is released. A reaction at 37 °C for 180 min yields N2-allyl-m7GpppA (3 b, up to 90 %). b) The product 3 b together with tetrazole 4 or 5 is irradiated at 254 nm for 5 min and left at 4 °C o/n. The 1,3-dipolar cycloaddition reaction yields fluorescent pyrazoline cycloadduct 6 or 7 (yields not determined).
Mentions: In this work we established a chemo-enzymatic approach to fluorescently label the 5’ cap typical of eukaryotic mRNAs using a photoclick reaction (Scheme 2). The 5’ cap consists of an N7-methylated guanosine which is linked to the next nucleotide by a 5′–5′ triphosphate bridge. It is specifically recognized by trimethylguanosine synthases, which are RNA methyltransferases that transfer one or two additional methyl groups to the exocyclic N2.45–47 One such enzyme, GlaTgs2, is well characterized and transfers only a single methyl group.45,46

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.