Capped and gowned: A two-step approach can be used to site-specifically modify the 5'-cap of eukaryotic mRNAs. First, a trimethylguanosinesynthase variant recognizes the m(7)G cap structure and introduces bioorthogonal groups using S-adenosyl-L-methionine-based cosubstrates. Then, the enzymatically introduced reporter groups are further modified by thiol-ene or CuAAC click chemistry (see scheme).
The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox.
We present a chemo-enzymatic approach for site-specific labeling of 5'-capped RNAs based on bioorthogonal chemistry. A trimethylguanosine synthase was engineered to transfer a terminal azido moiety to the 5'-cap which could be further modified using strain-promoted azide-alkyne cycloaddition (SPAAC).
Compartmentalization
of single genes in water-in-oil emulsion droplets
is a powerful approach to create millions of reactors for enzyme library
selections. When these droplets are formed at ultrahigh throughput
in microfluidic devices, their perfect monodispersity allows quantitative
enzyme assays with a high precision readout. However, despite its
potential for high quality cell-free screening experiments, previous
demonstrations of enrichment have never been successfully followed
up by actual enzyme library selections in monodisperse microfluidic
droplets. Here we develop a three-step workflow separating three previously
incompatible steps that thus far could not be carried out at once:
first droplet-compartmentalized DNA is amplified by rolling circle
amplification; only after completion of this step are reagents for
in vitro
protein expression and, finally, substrate added
via picoinjection. The segmented workflow is robust enough to allow
the first
in vitro
evolution in droplets, improving
the protease Savinase that is toxic to
E. coli
for
higher activity and identifying a 5-fold faster enzyme.
Enzymatic transfer of 4-vinylbenzyl to the mRNA 5′-cap gives access to the fluorogenic photoclick and the inverse electron-demand Diels–Alder reaction.
The ability to detect and localize defined RNA strands inside living cells requires probes with high specificity, sensitivity, and signal-to-background ratio. To track low-abundant biomolecules, such as strands of regular mRNA, and distinguish fluorescence signal from the background after bioorthogonal reactions in cells, it is imperative to employ turn-on concepts. Here, we have presented a straightforward enzymatic approach to allow site-specific modification of two different positions on the 5' cap of eukaryotic mRNA with either identical or different small functional groups. The approach relies on two methyltransferases and analogues of their natural co-substrate, and it can be extended to a three-enzyme cascade reaction for their in situ production. Subsequent labeling by using bioorthogonal click reactions provided access to double labeling with identical fluorophores or dual labeling with two different reporter groups, as exemplified by a Cy5 dye, a FRET pair, and a fluorophore/biotin combination. Our dual-labeling strategy addresses the need for increased sensitivity and should improve the signal-to-background ratio after bioorthogonal reactions in cells.
Trimethylguanosine synthase from Giardia lamblia (GlaTgs2) naturally catalyzes methyl transfer from S-adenosyl-L-methionine (AdoMet) to the exocyclic N(2) atom of the 5'-cap--a hallmark of eukaryotic mRNAs. The wild-type enzyme shows substrate promiscuity and can also use the AdoMet-analog AdoPropen for allyl transfer. Here we report on engineering GlaTgs2 to enhance the activity on AdoPropen. A mutational analysis, involving an alanine scan of 10 residues located around the active site, was performed. Positions V34 and S38 were identified as mutational hot spots and analyzed in greater detail by testing NNK libraries. Kinetic analysis and thermostability measurements revealed V34A as the best variant of GlaTgs2, with a ∼10-fold improved specificity for AdoPropen. Double mutants did not yield additional improvements due to low catalytic efficiencies and thermal destabilization. Homologous Tgs enzymes from Homo sapiens and G. intestinalis were also investigated regarding their catalytic activity on AdoPropen. While neither the human wild-type (WT) enzyme nor any of its variants showed activity on AdoPropen, the homologue from G. intestinalis (GinTgs) was remarkably active on AdoPropen. Introducing the best substitution at the homologous position led to variant T34A with ∼40-fold higher specificity for AdoPropen than the original GlaTgs2 WT.
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