ИСТИНА

Molecular beacons (MB) are valuable tools in molecular biology, clinical diagnostics and analytical chemistry. The extraordinary selectivity of MBs for SNPs in complementary nucleic acids is based on the equilibrium between closed and opened forms. This equilibrium can be tuned by sequence changes in the stem and loop structures, improving either sensitivity or specificity of SNP detection. However, this approach is not universal and in some cases worsens SNP discrimination by MBs due to nonspecific target recognition. Another common problem in MB design is the formation of multiple secondary structures that compete with each other, including disruption of the stem-loop structure (Figure 1). In some cases, this leads to the increase of background fluorescence or to poor performance of the probes due to the participation of neighboring nucleotides from the loop in the stem formation. In this study we have demonstrated that incorporation of inexpensive nucleotide or non-nucleotide linkers between stem and loop regions in molecular beacons during automated oligonucleotide synthesis significantly improve their specificity and performance in SNP discrimination. Incorporation of linkers dissects stem and loop regions in MBs, eliminates some non-specific internal interactions and improves the quality of the analysis. We demonstrated that such modified molecular beacons outperform classical MBs for ADRB2, NOS3 and Factor V SNPs. Analysis by scatterplot of clinical samples demonstrated that these MBs are more robust for the determination of A/G substitutions than classic MBs. Previously developed approaches13 that restrict undesired stem interactions were focused mainly on the decrease of stem invasion or interactions of the stems with DNA targets. Here, we developed an approach that not only solves the problem of stem invasion due to independent thermodynamic behavior of hexaethylene glycol separated parts, but also exclude stem-loop interactions, which increase background and decrease efficiency of the assay. Excellent SNP selectivity, simple synthesis and moderate cost of the MBs developed in this study exceed previously developed MBs. There is no doubt that our approach is applicable not only to fluorescently labeled MBs, but also to other biosensors based on the use of nucleic acids with stem-loop structures.