Fig. 5.

Monitoring the DcpS-catalyzed enzymatic hydrolysis of unlabeled (m⁷GpppG) and Mant-labeled (Mant-m⁷GpppG) cap analogues by spectrofluorymetry. (A). Unlabeled m⁷GpppG is cleaved by DcpS to m⁷GMP and GDP. The fluorescence intensity of the sample observed at the wavelength characteristic for m⁷G moiety (ex.=260 nm, em.=360 nm) increases along with reaction progress because in the dinucleotide (m⁷GpppG) fluorescence of m⁷G is partially quenched by intramolecular stacking between m⁷G and G. The fluorescence changes were sufficient to monitor reaction progress at concentration of 1 μM or higher(B) Mant-m⁷GpppG (2) is cleaved by DcpS to Mant-m⁷GMP and GDP. The fluorescence intensity of the sample observed at the wavelength characteristic for Mant moiety (ex.=361 nm, em.=446 nm) decreases along with reaction progress because the fluorescence of Mant in dinucleotide is enhanced by intramolecular interactions with hydrophobic parts of the second nucleoside (guanosine). The fluorescence changes were sufficient to monitor reaction progress at concentration of 50 nM or higher, i.e. 20-fold lower than for unlabeled m⁷GpppG. The DcpS concentration was 20 nM (experiment with m⁷Gp₃G) or 200 nM (experiment with Mant-m⁷GpppG).