*3.3. Conjugation of ASOs with Vitamin B<sup>12</sup>*

The 50 -azide-B<sup>12</sup> was synthesized from commercially available vitamin B<sup>12</sup> as described by Chromi ´nski et al. [20]. Briefly, the 50 -hydroxy group of the B<sup>12</sup> was transformed into a good leaving group (a mesyl group), and subsequently, azidation reaction provided the desired 50 -azide-B12. The 5<sup>0</sup> position was chosen to avoid obstruction of both components of the conjugate [6]. The azide-B<sup>12</sup> was isolated through precipitation. MS and NMR data were in accordance with the reported data [20].

Each Cy3-labeled ASO-BCN, dissolved in Milli-Q water, was added to a solution of azido-B12, dissolved in DMSO (ASO: azido-B<sup>12</sup> = 1:2 equivalent). The resulting solution was transferred to a Biotage microwave reaction vial (0.5 mL) and sealed under a nitrogen atmosphere. The reaction was carried out on a Biotage Initiator microwave synthesizer at 60 ◦C for 3 h, whereupon all solvents were removed in vacuo, and the residue was re-dissolved in Milli-Q water (Figure 1b). Analytical RP-HPLC and MALDI-TOF MS were performed. The resulting solutions were de-salted by precipitation of the products by first adding an aqueous solution of sodium acetate (3 M, 15 µL) followed by the addition of

cold ethanol (1 mL, 99% *w*/*w*; −20 ◦C). The resulting suspensions were stored at −20 ◦C for 1 h, and after centrifugation (16,000× *g*, 5 min, 4 ◦C), the supernatants were removed, and the pellet further washed with cold ethanol (2 × 1 mL; −20 ◦C), dried for 2 h and then dissolved in Milli-Q water (1 mL). Mass spectra of B12-ASO conjugates were recorded using MALDI-TOF MS, and the purity was confirmed by analytical RP-HPLC. Concentrations of purified conjugates were determined by ultraviolet absorbance at 260 nm.

The same procedure was conducted to obtain fluorescently labeled B<sup>12</sup> (without ASO) where DBCO-sulfo-Cy3, instead of the Cy3-labeled ASOs, was conjugated to B<sup>12</sup> through click-chemistry.

## *3.4. Bacterial Strain and Growth Conditions*

*E. coli* K12 MG1655 was used in this study. To prepare the inoculum, the strain was grown overnight in tryptic soy broth (TSB) at 37 ◦C with shaking (180 rpm). To monitor both the inhibition of the *acpP* gene expression and the location of the conjugates, *E. coli* K12 was grown in Davis minimal medium at 37 ◦C with shaking (180 rpm) [49]. This medium lacks B<sup>12</sup> in its composition, which was crucial to ensure that the internalized B<sup>12</sup> comes from the control/conjugates incubated with bacteria [6,21].

#### *3.5. Bacterial Susceptibility Tests*

The inhibition of the expression of the essential *acpP* gene by the conjugates B12- ASOgapmer and B12-ASOsteric was evaluated by monitoring the growth of *E. coli* K12, using a standard microdilution method. An overnight culture of *E. coli* K12 was diluted to an OD<sup>600</sup> of 0.1 in fresh Davis minimum medium. These cell suspensions were added to wells of sterile 96-well plates and incubated with different concentrations of the tested compounds at 37 ◦C. The final concentration of the B12-ASOs and respective controls (B12, ASOgapmer, and ASOsteric) was 30 µM. As a control for the ASOs activity, the most well-studied vector-ASO conjugate was tested. In brief, a conjugate composed of the cell-penetrating peptide (KFF)3K and peptide nucleic acid (PNA) (Eurogentec, Seraing, Belgium), designed to hybridize with the same *acpP* sequence was tested in the same conditions as the B<sup>12</sup> conjugates. The absorbance at 600 nm was determined on a BMGLabtech SPECTROstar Nano microplate reader for 24 h. *E. coli* in medium without any added compound was used as control (CB). Experiments were performed in three independent biological replicates.
