Can Vitamin B12 Assist the Internalization of Antisense LNA Oligonucleotides into Bacteria?

Abstract

The emergence of bacterial resistance to traditional small-molecule antibiotics is fueling the search for innovative strategies to treat infections. Inhibiting the expression of essential bacterial genes using antisense oligonucleotides (ASOs), particularly composed of nucleic acid mimics (NAMs), has emerged as a promising strategy. However, their efficiency depends on their association with vectors that can translocate the bacterial envelope. Vitamin B₁₂ is among the largest molecules known to be taken up by bacteria and has very recently started to gain interest as a trojan-horse vector. Gapmers and steric blockers were evaluated as ASOs against Escherichia coli (E. coli). Both ASOs were successfully conjugated to B₁₂ by copper-free azide-alkyne click-chemistry. The biological effect of the two conjugates was evaluated together with their intracellular localization in E. coli. Although not only B₁₂ but also both B₁₂-ASO conjugates interacted strongly with E. coli, they were mostly colocalized with the outer membrane. Only 6-9% were detected in the cytosol, which showed to be insufficient for bacterial growth inhibition. These results suggest that the internalization of B₁₂-ASO conjugates is strongly affected by the low uptake rate of the B₁₂ in E. coli and that further studies are needed before considering this strategy against biofilms in vivo.

Keywords: 2′OMe; LNA; antibacterial drug; antisense oligonucleotides; nucleic acid mimics; vitamin B12.

Figure 1

Different mechanisms that play a role in the modulation of the RNA function in bacteria. (a) Upon hybridization of a gapmer (red), the RNase H is recruited, and the target is degraded. (b) Steric hindrance of the ribosome caused by the hybridization between a steric blocker (red) and the complementary mRNA sequence. Figure created using BioRender.

Figure 2

B₁₂ and antisense oligonucleotides (ASOs) conjugation: sequences and structures. (a) Sequence of the ASO_gapmer and the ASO_steric (LNA nucleotide monomers are represented with upper case letters preceded by l, 2′OMe monomers are represented with upper case letters preceded by m, and DNA monomers are represented by lower case letters) and structure of 5′-end azide-modified B₁₂ used in this study. The arrow points to the region of conjugation. (b) Schematic illustration of the synthesis of the B₁₂-ASO conjugates through copper-free azide-alkyne chemistry.

Figure 3

Growth of E. coli K12 in Davis minimal medium supplemented with B₁₂-ASO_gapmer, B₁₂-ASO_steric, and B₁₂ (at a concentration of 30 μM). CB represents the bacterial growth control in medium without any supplementation. Growth inhibition of E. coli K12 using a cell-penetrating peptide conjugated with an ASO composed of peptide nucleic acids (PNAs) (cell-penetrating peptides (CPP)-PNA) at a concentration of 30 μM is also shown. Results from three independent experiments (using duplicates in each) are presented as mean values and respective standard deviations. Statistical differences are indicated when appropriate in * (p ≤ 0.0001, ****).

Figure 4

Interaction of Cy3-labeled ASOs, B_12, and B₁₂ conjugates (concentration of 30 μM) with E. coli K12 after 4 h. Bacteria are counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Images are representative of three independent experiments (using duplicates in each). Scale bar represents 5 μm.

Figure 5

Cellular localization of B₁₂ conjugates, B_12, and DAPI control in E. coli K12. B₁₂ conjugates and B₁₂ are mainly found on the OM, while the DAPI control is mostly associated with the cytosol. No significant differences were observed between the different internalized conjugates and between the conjugates and the B₁₂ control (p > 0.05). Significant differences were observed between the membrane and cytosol-associated compounds (p ≤ 0.0001, ****). The fluorescence of each fraction present in the DAPI control is significantly different from the tested counterparts (p ≤ 0.0001). Results are presented as mean values and respective standard deviation from three independent assays (using duplicates in each).

Figure 6

Fractionation protocol adapted from Bandula et al. [50]. A series of washing steps with a Triton X-100 gradient allows the isolation of the membrane and the cytosol fractions.