Assessment of methodologies based on the formation of antiparallel triplex DNA structures and fluorescent silver nanoclusters for the detection of pyrimidine-rich sequences

In this work, strategies for the detection of pyrimidine-rich DNA target sequences based on the formation of duplex and antiparallel triplex structures are studied. The presence of the target is detected from the changes in fluorescence of silver nanoclusters stabilized by the corresponding complementary DNA probes. In all cases, the formation of intermolecular structures has been assessed by means of melting experiments and multivariate analysis. In the case studied, it has been observed that the formation of antiparallel triplex structures produces changes in fluorescence properties that could be more useful for analytical purposes than those observed when only duplex structures are formed. In particular, the use of silver nanoclusters confined within a loop rich in cytosine-type bases in the antiparallel triplex structure resulting from the interaction of probe and analyte has been shown to produce an increase in red fluorescence. This latter probe has been shown to be selective against target sequences that have mismatches that could affect the formation of stable duplex structures, while it has been shown to tolerate a small number of purine mismatches that could affect the stability of the resulting antiparallel triplex structure. As a final remark, it should be noted that this methodology could also be used in the development of analytical procedures that allow the detection of antiparallel triplex structures, which are difficult to observe with other spectroscopic methods.