As demonstrated above, the DNA/miRNA hybrids can
form a triplex structure with PMNT molecules, which has a
maximum absorption at 400 nm and shows a yellow color.
When the miRNA of DNA/miRNA hybrids is digested by
RNase H, the released ssDNA combines with PMNT mole-
cules to form DNA/PMNT duplex, where a marked red-shift
of the maximum absorption can be observed (l max = 525 nm)
and the solution becomes red. Fig. 4 shows the absorption
spectra of PMNT as a function of the RNase H digestion time.
In these experiments, the RNase H (0.3 Units) is added to the
complementary DNA/miRNA solution. After a specific incu-
bation period, PMNT is added in and then the absorption
spectrum is immediately measured. Gradual color changes of
the solution from yellow to red are observed along with the
RNase H digestion time from 0 to 25 min. As shown in
Fig. 4A, the absorption maximum of PMNT at 525 nm is
gradually increased and that at 400 nm is gradually decreased
with increasing digestion time. The absorbance ratio of A 525
nm /A 400 nm is linearly dependent on the digestion time in the
period from 0 to 15 min (Fig. 4B). The results prove that the
absorption and color changes produced by PMNT conforma-
tion changes can be applied to detect RNase H activity with
rapid processes and a simple instrument.