Figure 1.
Schematic representation of the pyrene-4-maleimide synthesis and structure.
A. Pyrene maleimide structure. B. Pyrene-4-maleimide synthesis route and structure. Ph3P: triphenylphosphine; DIAD: diisopropyl azodicarboxylate.
Figure 2.
Monomer and excimer emission in the presence of organic compounds.
A. Pyrene maleimide. B. Pyrene-4-maleimide. C. Pyrene maleimide vs. pyrene-4-maleimide in mercaptoethanol. D. Pyrene maleimide vs. pyrene-4-maleimide in butanethiol. The concentrations of the fluorescent probes and organic compounds were 3 µM and 1 mM, respectively. The labels in panels A and C also apply to panels B and D, respectively. Peaks 1–4 are indicated (see text). Data in panels A–C were normalized to peak 1 intensity in mercaptoethanol. Data in panel D were normalized to peak 4 intensity in butanethiol.
Figure 3.
Emission of pyrene compounds reacted with thiol-modified DNA.
A. Emission spectra of single-stranded DNA containing a 5′ end thiol group (DNA14) reacted with pyrene-4-maleimide. Data were normalized to peak 1 intensity from reduced DNA14. [DNA14] was 0.5 µM, and [pyrene-4-maleimide] was 2 µM. B. Emission spectra of double-stranded DNA reacted with pyrene maleimide. DNA14-14c: DNA14 annealed to fully complementary DNA with a 3′ end thiol group (DNA14c). DNA14-12c: DNA14 annealed to a 3′ end two-base shorter complementary oligonucleotide with a 3′ end thiol group. DNA14-10c: DNA14 annealed to a 3′ end four-base shorter complementary oligonucleotide with a 3′ end thiol group. C. Emission spectra of double-stranded DNA reacted with pyrene-4-maleimide. Insert: schematic representation of the experimental system showing the double-stranded DNAs labeled with pyrene-4-maleimide. The pyrenes are represented by green rhomboids. Data in panels B and C were normalized to peak 1 intensity. The labels in panel B also apply to panel C. D. Excimer/monomer emission ratio. The values were calculated as: excimer/monomer = Ipeak 4/Ipeak 1, where I is the highest intensity of the peak, and peak 1 and peak 4 correspond to the excimer and monomer emission peaks. Averages ± SEM from experiments such as those shown in panels B and C (n = 3 for pyrene maleimide, and n = 4 for pyrene-4-maleimide). The asterisk denotes P<0.05 for the pyrene-4-maleimide DNA14-14c adduct vs. each of the other adducts presented in panel D. The concentrations of the fluorescent probes and double-stranded DNA were 4 µM and 0.5 µM, respectively.
Figure 4.
Double-stranded DNA models with pyrene maleimides attached via thiol linkers.
A. Stick representation view along the DNA long axis. Pyrene-4-maleimide attached to DNA14-14c and DNA14-12c is shown in green and red, respectively. Pyrene maleimide attached to DNA14-14c is shown in blue. The 5′ thiol was present in the 14-bp long strand in all cases. The 3′ thiol was present in the 14-bp or 12-bp long complementary strands. B. Stick representation view perpendicular to that in panel A. Only the pyrenes are shown for clarity. Color coding as in panel A. See Materials and Methods for details.
Figure 5.
Long lifetime of pyrene-4-maleimide excimer emission.
DNA14: reduced single-stranded DNA14 adduct. DNA14-14c: double-stranded DNA14-14c adduct. IRF: instrument response function. The red lines are fits of the data to multi-exponential functions, with the two weighted residuals (Ri) vs. time plots corresponding to the double-stranded (top) and single-stranded (bottom) data fits.