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.
Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy has remained challenging to date. We here demonstrate an efficient and generally applicable posttranscriptional SDSL method for large RNAs using an expanded genetic alphabet containing the NaM-TPT3 unnatural base pair (UBP). An alkyne-modified TPT3 ribonucleotide triphosphate (rTPT3COTP) is synthesized and site-specifically incorporated into large RNAs by in vitro transcription, which allows attachment of the azide-containing nitroxide through click chemistry. We validate this strategy by SDSL of a 419-nucleotide ribonuclease P (RNase P) RNA from Bacillus stearothermophilus under non-denaturing conditions. The effects of site-directed UBP incorporation and subsequent spin labeling on the global structure and function of RNase P are marginal as evaluated by Circular Dichroism spectroscopy, Small Angle X-ray Scattering, Sedimentation Velocity Analytical Ultracentrifugation and enzymatic assay. Continuous-Wave EPR analyses reveal that the labeling reaction is efficient and specific, and Pulsed Electron-Electron Double Resonance measurements yield an inter-spin distance distribution that agrees with the crystal structure. The labeling strategy as presented overcomes the size constraint of RNA labeling, opening new avenues of spin labeling and EPR spectroscopy for investigating the structure and dynamics of large RNAs.
