Vitamin B12 transports modified RNA into E. coli and S. Typhimurium cells.

Specifically designed, antisense oligonucleotides are promising candidates for antibacterial drugs. They suppress the correct expression of bacterial genes by complementary binding to essential sequences of bacterial DNA or RNA. The main obstacle in fully utilizing their potential as therapeutic agents comes from the fact that bacteria do not uptake oligonucleotides from their environment. Herein, we report that vitamin B12 can transport oligonucleotides into Escherichia coli and Salmonella typhimurium cells. 5'-Aminocobalamin with an alkyne linker and azide-modified oligonucleotides enabled the synthesis of vitamin B12-2'OMeRNA conjugates using an efficient "click" methodology. Inhibition of protein expression in E. coli and S. Typhimurium cells indicates an unprecedented transport of 2'OMeRNA oligomers into bacterial cells via the vitamin B12 delivery pathway.

Conformational Dynamics of Cyanocobalamin and Its Conjugates with Peptide Nucleic Acids.

Vitamin B12 also called cobalamin (Cbl) is an important enzymatic cofactor taken up by mammalian and also by many bacterial cells. Peptide nucleic acid (PNA) is a synthetic DNA analogue that has the ability to bind in a complementary manner to natural nucleic acids. Provided that PNA is efficiently delivered to cells, it could act as a steric blocker of functional DNA or RNA and regulate gene expression at the level of transcription or translation. Recently, Cbl has been examined as a transporter of various molecules to cells. Also, PNA, if covalently linked with Cbl, can be delivered to bacterial cells, but it is crucial to verify that Cbl does not change the desired PNA biological properties. We have analyzed the structure and conformational dynamics of conjugates of Cbl with a PNA monomer and oligomer. We synthesized a cyanocobalamin derivative with a PNA monomer C connected via the triazole linker and determined its NMR spectra. Using microsecond-long molecular dynamics simulations, we examined the internal dynamics of cyanocobalamin-C, its conjugate with a 14-mer PNA, and free PNA. The results suggest that all compounds acquire rather compact structures but the PNA oligomer conformations vary. For the Cbl-C conjugate the cross-peaks from the ROESY spectrum corroborated with the clusters from molecular dynamics trajectories. Within PNA the dominant interaction is stacking but the stacking bases are not necessarily neighboring in the PNA sequence. More bases stack in free PNA than in PNA of the conjugate, but stacking is less stable in free PNA. PNA in the conjugate is slightly more exposed to solvent. Overall, cyanocobalamin attached to a PNA oligomer increases the flexibility of PNA in a way that could be beneficial for its hybridization with natural nucleic acid oligomers.