We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI·P(OEt) as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-GalNAc alkyne gives oligonucleotide-GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high-throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2'-modified siRNA.

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For the first time we used CuAAC click reaction for the synthesis of cyanine labeled qPCR probes. We performed the comparison of dyes and quenchers (Cy5 vs disulfo-Cy5 and BHQ2 vs BHQ3) in several TaqMan probes with different stability of the secondary structure. Based on the studies of thermal quenching of dyes, increase of fluorescence during probe melting and in the course of qPCR, we suggest that disulfo-Cy5-BHQ2 pair is a preferable one for molecular beacons due to the highest increase of fluorescence during melting. As Cy5-BHQ3 pair gave better Cq and maximal relative increase of fluorescence in qPCR in comparison to others we recommend this pair for Taqman style probe design.

Molecular beacons (MBs) are valuable tools in molecular biology, clinical diagnostics and analytical chemistry. Here we describe a novel approach for the design of MBs with nucleotide or non-nucleotide linkers between the stem and loop regions. Such modified MBs have significantly improved specificity and performance for single nucleotide polymorphism (SNP) detection. These advantages are especially distinct, when compared to the classic MBs, in the case of possible interactions between the stem and loop regions. We demonstrated the applicability of such modified MBs for the discrimination of common Factor V, NOS3 and ADRB2 SNPs in model plasmids and in clinical samples. The developed approach could be applicable not only to fluorescently labeled MBs, but also to other biosensors based on nucleic acids with stem-loop structures.