Oligonucleotide conjugates with boron clusters have found applications in different fields of molecular biology, biotechnology, and biomedicine as potential agents for boron neutron capture therapy, siRNA components, and antisense agents. Particularly, the closo-dodecaborate anion represents a high-boron-containing residue with remarkable chemical stability and low toxicity, and is suitable for the engineering of different constructs for biomedicine and molecular biology. In the present work, we synthesized novel oligonucleotide conjugates of closo-dodecaborate attached to the 5'-, 3'-, or both terminal positions of DNA, RNA, 2'-O-Me RNA, and 2'-F-Py RNA oligomers. For their synthesis, we employed click reaction with the azido derivative of closo-dodecaborate. The key physicochemical characteristics of the conjugates have been investigated using high-performance liquid chromatography, gel electrophoresis, UV thermal melting, and circular dichroism spectroscopy. Incorporation of closo-dodecaborate residues at the 3'-end of all oligomers stabilized their complementary complexes, whereas analogous 5'-modification decreased duplex stability. Two boron clusters attached to the opposite ends of the oligomer only slightly influence the stability of complementary complexes of RNA oligonucleotide and its 2'-O-methyl and 2'-fluoro analogs. On the contrary, the same modification of DNA oligonucleotides significantly destabilized the DNA/DNA duplex but gave a strong stabilization of the duplex with an RNA target. According to circular dichroism spectroscopy results, two terminal closo-dodecaborate residues cause a prominent structural rearrangement of complementary complexes with a substantial shift from the B-form to the A-form of the double helix. The revealed changes of key characteristics of oligonucleotides caused by incorporation of terminal boron clusters, such as the increase of hydrophobicity, change of duplex stability, and prominent structural changes for DNA conjugates, should be taken into account for the development of antisense oligonucleotides, siRNAs, or aptamers bearing boron clusters. These features may also be used for engineering of developing NA constructs with pre-defined properties.
Cancer is a major health risk in the modern society that requires rapid, reliable, and inexpensive diagnostics. Because of the low abundance of cancer DNA in biofluids, current detection methods require DNA amplification. The amplification can be challenging; it provides only relative quantification and extends time and cost of an assay. Herein, we report a new oligonucleotide hybridization platform for amplification-free detection of human cancer DNA. Using a large PEG-capture probe allows rapid separation of the bound (mutant) versus unbound (wild type) DNA. Next, a supramolecular hydrogel forming peptide attached to a detection oligonucleotide probe serves as a signal amplification tool. Having screened multiple short peptides and fluorophores, we identified the system P1 + cyanine 3.5 that allows for sensitive quantitative detection of mutation L858R in EGFR oncogene. The peptide-fluorophore-based assay provides absolute target DNA quantification at the detection limit of 20 ng cancer DNA versus >500 ng for Cy3.5-labeled oligonucleotide in only 1 hour.
