Conformational Dynamics of Cyanocobalamin and Its Conjugates with Peptide Nucleic Acids

Abstract

Vitamin B 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.

Does a Conjugation Site Affect Transport of Vitamin B12–Peptide Nucleic Acid Conjugates into Bacterial Cells?

“…Most effective is a conjugate with PNA anchored at the R 5′ ‐OH group, whereas modifications at the peripheral c , e , and meso positions adversely affect the uptake; thus indicating the importance of c‐ and e ‐amides during the recognition process. The PNA forms compact structures in solution, but its flexibility (required for hybridization with RNA) increases upon the attachment of vitamin B 12 at the R 5′ ‐OH position, as previously found by us in molecular dynamics simulations . However, the structural dynamics of PNA attached at other vitamin B 12 positions is yet to be elucidated.…”

Antibacterial Peptide Nucleic Acids—Facts and Perspectives

“…In recent years, PNA was combined with vitamin B 12 using different linkers. Vitamin B 12 was also found to improve PNA solubility and make the PNA in the conjugate adopt a more extended conformation in comparison with free single-stranded PNA [124]. Furthermore, we have shown that vitamin B 12 acts as a carrier of PNA to E. coli and Salmonella enterica subsp.…”

Advances in therapeutic bacterial antisense biotechnology

“…Screening of further bacterial mRNA virulence targets and assessment of optimal ASO lengths to balance specificity and bacterial cell entry are also additional areas for study. Recently the fact that some bacteria are capable of vitamin B12 uptake was exploited to achieve enhanced PNA delivery into S. typhimurium and E. coli by a novel conjugation of PNA antisense oligomers to vitamin B12 (Pieńko et al 2017; Równicki et al 2017). These PNA-B12 conjugates were synthesized by a Cu-catalyzed 1,3-dipolar cycloaddition of the vitamin B12 azide to PNAs possessing a terminal alkyne group.…”