  We proposed a dual-template multi-cycled DNA nanomachine driven by polymerase
nicking enzyme with high efficiency. The reaction system simply consists of two
templates (T1, T2) and two enzymes (KF polymerase, Nb.BbvCI). The two templates
are similar in structure (X-X-Y, Y-Y-C): primer recognition region, primer
analogue generation region, output region (3 to 5), and there is a nicking site
between each two regions. Output of T1 is the primer of T2 and G-rich fragment
(G3) is designed as the final products. In the presence of HIV-1, numerous of
G3 were generated owing to the multi-cycled amplification strategy and formed
into G-triplex ThT complex after the addition of thioflavin T (ThT), which
greatly enhanced the fluorescence intensity as signal reporter in the
label-free sensing strategy. A dynamic response range of 50 fM-2 nM for HIV-1
gene detection can be achieved through this multi-cycled G-triplex machine, and
benefit from the high efficiency amplification strategy, enzymatic reaction can
be completed within 45 minutes followed by fluorescence measurement. In
addition, analysis of other targets can be achieved by replacing the template
sequence. Thus there is a certain application potential for trace biomarker
analysis in this strategy.

Quantitative Methods