Chemo-enzymatic approaches provide powerful tools for labeling distinct biomolecules specifically, even within living cells.
Apart from proteins and DNA also tRNA and pre-mRNAs have been modified successfully.
In our group we established a chemo-enzymatic strategy for specific modification of eukaryotic mRNAs. For this purpose, we made use of a Tgs variant, capable of transferring alkenyl- or alkynyl- residues specifically to the mRNA cap. The enzymatically labeled structure was then amenable to modification by different click reactions, such as thiol-ene or CuAAC.
This strategy could provide a novel basis for modifying eukaryotic mRNAs in living cells, making this species accessible for isolation or visualization.
Methods (from Supplement)
Enzymatic modification of cap-analogue m7GpppA
For enzymatic modification the cap analogue m7GpppA (1 mM) was incubated with AdoPropen (0.5-1 mM) respectively AdoEnYn (360-740 µM) in presence of GlaTgs2 or variant (5-120 µM) as well as 4 µM MTAN and 3 µM LuxS in PBS (pH 7.4) or PUS buffer (without DTT) at 37 °C for 3 hours. Reactions for subsequent use in TEC were stopped by adding 1/10 volume 1 M HClO4, those for CuAAC were stopped by heating samples for 15 min at 68 °C, followed by dialysis against PUS buffer (with 5 mM DTT) for 15 min. Enzymatic conversions were analyzed by HPLC after protein precipitation with 1 M HClO4 on an analytical NUCLEODUR® C18 Pyramid (5 μm, 125×4 mm).
Thiol-Ene Click reaction
In situ generated N2-allyl-m7GpppA was used for TEC after protein precipitation without any further purification. Samples were degassed with argon before biotin-thiol 6 (40 mM) and VA-044 (800 µM) were added. Reactions were performed for up to 8 h at 44 °C under exclusion of oxygen. Afterwards samples were analyzed by reversed-phase HPLC.
CuAAC with RNA
RNA is generated by in vitro transcription using T7 polymerase, purified by phenol:chloroform extraction and precipitated using 2-propanol. RNA is resuspended in PBS and concentration is determined by in-gel quantification. For our experiments RNA solutions with about 19 µg/µL worked best.
Capping of transcribed RNA is achieved with the vaccinia capping system (NEB) In detail, 0.9 nmol RNA were allowed to react with GTP (10 nmol) and vaccinia capping enzyme (20 U) with or without AdoMet (2 nmol) in a final volume of 20 µL for 1 h at 37 °C. Make sure to use the correct concentration of RNA in this step. Afterwards samples were heated for 15 min at 65 °C and incubated 15 min at 4 °C with cation exchanger P11 cellulose phosphate (Whatman). Supernatant was precipitated and pellet was resuspended in GlaTgs2-Var1 (37-49 µM), MTAN (1.2 µM), LuxS (0.6 µM, all purified using an ÄKTA purifier™ system and HisTrap™ FF 1 columns (GE Healthcare)), AdoEnYn (2, 1.5-1.7 mM) in the presence of 0.25 µL RiboLock RNase Inhibitor (Thermo Scientific) and adjusted to a final volume of 5 µL with PUS buffer. If labeling is not successful it can help to increase AdoEnYn concentration in this step and to make sure that labeling efficiency with GlaTgs variant is sufficient using a cap-analogue as substrate.
Samples are then incubated for 90 min at 37 °C, RNA is precipitated and used for CuAAC. For this, RNA is resuspended in PUS-buffer containing 5 mM DTT (4 µL), 3.2 µL Click solution (DMSO:tBuOH 3:1; Jenaobioscience) is added as well as 0.8 µL of a 2.5 mM Eterneon-azide solution CuBr (stored in a glovebox) is solved in Click solution to give a concentration of 300 mM and immediately diluted in a 111 mM TBTA to give a final CuBr concentration of 30 mM. The Cu/TBTA solution (1.2 µL) is added to the RNA, which is then incubated at 37 °C for 1 hour. Samples are analyzed on 10 % denaturing gels.
Rentmeister Group | University of Hamburg, Germany
Andrea Rentmeister is a Professor for Biochemistry at the Westfälische Wilhelms-Universität Münster. When the movie was produced, she was a Junior Professor for Biochemistry at the University of Hamburg and an Emmy Noether group leader. Research in her group focusses on novel methods to selectively label biomolecules in living cells. To achieve this, techniques from chemistry, molecular biology, and protein biochemistry are harnessed The main interest is labeling of mRNA, which can localize to distinct subcellular regions in various cell types ranging from bacteria to
Josephin M. Holstein studied chemistry at the University of Hamburg and is working as a PhD student in the Rentmeister group since 2012, focusing on the adaptation of SPAAC for labeling eukaryotic mRNAs.
Daniela Schulz studied chemistry at the University of Hamburg, where she started her PhD thesis in 2010 with the aim to establish a chemo-enzymatic approach for labeling eukaryotic mRNAs.
We thank the “Sea Life Deutschland GmbH” for the opportunity to film at the SEA LIFE Timmendorfer Strand.
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