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

Maciej Giedyk a, Agnieszka Jackowska a, Marcin Równicki bc, Monika Kolanowska bd, Joanna Trylska *b and Dorota Gryko *a
aInstitute of Organic Chemistry Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland. E-mail: dorota.gryko@icho.edu.pl
bCentre of New Technologies University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland. E-mail: joanna@cent.uw.edu.pl
cCollege of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland
dGenomic Medicine, Medical University of Warsaw, Banacha 1a, 02-097 Warsaw, Poland

Received 25th June 2018 , Accepted 19th November 2018

First published on 19th November 2018


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.


The routine use and, most often, overuse of antibiotics has led to the rapid development of bacterial resistance.1 As a consequence, new antibacterial agents are urgently needed and to this end, antisense oligonucleotides inhibiting gene expression in a sequence-specific way at the translational level are promising candidates.2,3 However, bacteria do not uptake oligonucleotides from their environment, which is the limiting factor in their potential use as antibiotics.4 Though a variety of vectors including lipids,5 polymers,6 cell penetrating peptides,7 and nanoparticles8 have been explored, the combination of hydrophobic phospholipid tails from the cell membrane and the lack of an endocytosis mechanism impedes their effective transport into the bacteria's interior.9 Conjugation of cell penetrating peptides to uncharged nucleic acid analogues (e.g. peptide nucleic acids, PNAs) facilitates delivery to various bacterial cells.10 But for the charged oligonucleotides, even for 2′OMeRNA, only heat shock11 and electroporation12 allow their introduction into bacterial cells. Thus an appropriate delivery system is yet to be discovered.13

The use of vitamin B12 (cobalamin, 1, Fig. 1) as a drug delivery vehicle into eukaryotic cells is well documented and as such increases the bioavailability of different therapeutics.14


image file: c8cc05064c-f1.tif
Fig. 1 Vitamin B12 (1).

Not all bacteria produce vitamin B12 (1), therefore they develop specific mechanisms for its uptake from their environment.15 Prokaryotic cells actively transport vitamin B12 (1) using a cascade of membrane proteins.16 In Escherichia coli and Salmonella enterica subsp. enterica serovar Typhimurium, vitamin B12 (1) is recognized by a number of bacterial receptors, responsible for its passage into the cytoplasm.17,18

As one of the principal difficulties relating to the application of antisense oligonucleotides as therapeutic agents for bacterial infections concerns their penetration into bacteria, we investigated if vitamin B12 can transport them into Gram-negative bacterial cells via its specific mechanism. Herein, we present the unprecedented delivery of antisense 2′OMeRNA oligomers into E. coli and S. Typhimurium cells using vitamin B12 (1) confirmed by observing inhibition of the expression of the red fluorescent protein (RFP) in bacterial cells by sequence-specific 2'OMeRNA.

The most common methods for the synthesis of antisense oligonucleotides are based on the solid phase synthesis of RNA possessing reactive nucleotide analogues followed by their post-synthetic functionalization. Along this line, we envisaged that 2′OMeRNA and vitamin B12 could be conjugated via the CuAAC (copper-catalysed azide–alkyne cycloaddition)19 reaction. Such a strategy allows for a robust connection between the two components via the amide bond and the triazole ring (Fig. 2).


image file: c8cc05064c-f2.tif
Fig. 2 Synthesis of 2′OMeRNA–vitamin B12 conjugates via copper(I)-catalysed alkyne–azide cycloaddition (CuAAC).

We have chosen the 5′-position as the conjugation site since our recent work on the delivery of peptide nucleic acid – vitamin B12 conjugates into prokaryotic cells shows that neither molecule obstructs each other.20 Moreover, modification of vitamin B12 at this position gives access to the amino-cobalamin (5′-NH2), a useful tool for attaching biologically active compounds via the amide bond.21 Its reaction with 6-heptynoic acid gave vitamin B12 derived alkyne 2 suitable for conjugation with azides using the copper-catalysed alkyne–azide cycloaddition reaction (Scheme 1). We have chosen this reaction because it has been widely applied in the functionalization of RNA and RNA components.22


image file: c8cc05064c-s1.tif
Scheme 1 Reaction of vitamin B12 derivative 2 with azides 3a–c. Conditions: alkyne 2 (20 mM), azide 3 (20 mM), Cu-TBTA (0.5 mM), L-ascorbic acid (0.5 mM), DMSO/buffer, pH = 7 (1[thin space (1/6-em)]:[thin space (1/6-em)]1), rt, and 48 h.

In the first step, we developed the synthesis of azides 3: deoxycytidine derivative 3a bearing the nucleophilic amino group, deoxycytidine derivative 3b with the negatively charged phosphate linker attached and 5′azidothymidine 3c (for the description of the synthesis of azides 3a–c, see the ESI). Because the 2′OMeRNA conjugation requires high efficiency in dilute solutions, conditions for the model reaction of alkyne 2 with azide 3a were optimised in terms of catalyst, reducing agent, solvent, and reaction time (for full optimisation tables, see the ESI). The reaction was performed in a water/DMSO mixture (1[thin space (1/6-em)]:[thin space (1/6-em)]1) to ensure full solubility of the reactants. The use of Cu-TBTA and L-ascorbic acid (AA) as a reducing agent proved the most effective affording conjugate 4a in high yields. In order to check if the charged phosphate or carbonyl group (present in commercial available oligonucleotides) influences the course of the CuAAC reaction, azides 3b and 3c were reacted with alkyne 2 under the developed conditions. Compounds 4b and 4c were obtained in 78% and 69% yields, respectively. With the optimised procedure in hand, the N3-modified 2′OMeRNA oligonucleotides 5a, 5b, and 5c were conjugated with vitamin B12 (Scheme 2).


image file: c8cc05064c-s2.tif
Scheme 2 Schematic representation of the synthesis of 2′OMeRNA–vitamin B12 conjugates via copper(I)-catalysed alkyne–azide cycloaddition (CuAAC). Conditions: (i): alkyne 2 (1 mM), azides 5a–c (0.5 mM), Cu-TBTA (1.5 mM), L-ascorbic acid (6 mM), DMSO/buffer pH = 7 (1[thin space (1/6-em)]:[thin space (1/6-em)]1), r.t., and 20 h. aConversion determined by HPLC.

The oligonucleotide sequence was designed as complementary to the mRNA transcript encoding RFP (specifically the Shine–Dalgarno and start codon region) to inhibit RFP production in E. coli and S. Typhimurium cells carrying the plasmid with the mrfp1 gene.20 Oligonucleotide azides 5a (5′-deoxythymidine-CAUCUAGUAUUUCU-3′), comprised an additional thymidine spacer, and 5b (5′-hexyl-CAUCUAGUAUUUCU-3′) with a hexyl linker, whereas analogue 5c (5′-UUUCUAGUCUCAUA-3′), a scrambled sequence oligonucleotide consisting of the same bases but not complementary to the targeted region, was designed as a control. The reaction of azide 5a with the vitamin B12 derivative 2 under the optimised CuAAC conditions proceeded selectively to give the desired product B12–2′OMeRNA 6a, but in low conversion (36% according to HPLC). This was rectified with the further addition of AA giving >97% conversion. After purification, conjugate 6a was analysed by ESI MS and the signal at m/z = 6318.50 corresponds to a pseudomolecular ion [M−H]. The same methodology was applied for azides 5b and 5c giving B12–hexyl-2′OMeRNA 6b (71% and purity 75% as compound decomposed during purification) and B12–2′OMeRNAscrambled conjugate 6c (80%) respectively.

In the next step, we tested the stability of the conjugates in a bacteria medium. Conjugates 6a–c were added at 50 μM concentrations into Davis Minimal Broth.23 Following overnight incubation at 37 °C with shaking, RP-HPLC analysis was performed. The resulting chromatograms did not show any detectable differences before and after incubation. Therefore, these conjugates were considered stable in the presence of the tested medium (see the ESI).

In order to determine if vitamin B12 transports 2′OMeRNA oligomers to bacteria, we monitored the inhibition of RFP production in E. coli and S. Typhimurium cells expressing RFP as previously described.20 In particular, the 2′OMeRNA complementary to the mrfp1 mRNA transcript, if delivered into bacterial cells, should block RFP expression. Thus, a decrease in red fluorescence of cells denotes the antisense effect and is a measure of transport efficiency. Indeed, after 20 h of incubation, the level of fluorescence from E. coli and S. Typhimurium treated with vitamin B12–2′OMeRNA 6a was reduced by approx. 50% (at concentrations of 1 μM and higher) as compared to the untreated growth control and the scrambled non-complementary oligonucleotide 6c (Fig. 3).


image file: c8cc05064c-f3.tif
Fig. 3 Relative fluorescence measured after the treatment of E. coli and S. Typhimurium cells with vitamin B12–2′OMeRNA conjugates 6a, 6c, and vitamin B12 only. Top graphs show data for finer concentrations in the 0–2 μM range and bottom graphs show coarser data for the 0.5 to 16 μM concentration range. Error bars represent standard errors from three independent biological experiments. The differences between the conjugate 6a and 6c, as well as 6a and vitamin B12 only, are significant with P ≤ 0.05 (determined using an ANOVA method without correction for multiple comparisons). Full data including conjugate 6b are in the ESI.

Therefore, for 6a the fluorescence decrease arose from the uptake of antisense 2′OMeRNA that was delivered by vitamin B12 and specifically targeted the mRNA transcript encoding RFP. In addition, the fluorescence intensity remains at the same level after adding free vitamin B12 (Fig. 3). Therefore, the inhibitory effect depends both on the 2′OMeRNA sequence and its conjugation to vitamin B12. This is the first reported case of 2′OMeRNA being delivered to bacterial cells using vitamin B12 as a carrier. Note that after the initial dose-dependence up to 1 μM, the RFU does not decrease further suggesting that the cellular vitamin B12 uptake reached saturation. In addition, in a similar experiment in a vitamin B12-rich medium, the uptake of the conjugate does not occur, presumably due to saturation of the receptors involved in the transport of vitamin B12.

To exclude unspecific toxicity of the conjugates 6a and 6c to bacterial cells, we monitored if they alone affected E. coli and S. Typhimurium growth. After 20 h of incubation we did not observe any significant changes in the optical density (at 600 nm) of the cultures. Furthermore, the cytotoxic effect of the 2′OMeRNA–B12 conjugates 6a and 6c on human embryonic kidney cells 293 (HEK 293) was also verified. HEK 293 cells were incubated with conjugates 6a and 6c for 48 h, with the highest concentration (16 μM) analysed on E. coli and S. Typhimurium, and no significant impact on HEK 293 cell-viability, when compared to the untreated control, was observed (Fig. 4). The cytotoxic test was performed using an MTT assay and measured at 590 nm.24


image file: c8cc05064c-f4.tif
Fig. 4 Cytotoxic effect of vitamin B12–2′OMeRNA conjugates 6a and 6c on HEK293, E. coli and S. Typhimurium cells. The results are presented as the percent of untreated control cells.

In summary, we present an efficient methodology for the preparation of stable vitamin B12–2′OMeRNA conjugates. A vitamin B12 derivative with an alkyne linker appended at the 5′ position was conjugated to 2′OMeRNA bearing the azide functionality via the copper(I)-catalysed alkyne–azide cycloaddition (CuAAC) reaction. Such conjugates penetrated into bacteria proving that vitamin B12 is capable of transporting 2′OMeRNA oligonucleotides into E. coli and S. Typhimurium cells.

This study has laid the foundation for future works on vitamin B12 as a non-invasive carrier of antisense RNA oligonucleotides into Gram-negative bacterial cells.

The financial support for this work was provided by the National Science Centre, Poland, grant SYMFONIA 2014/12/W/ST5/00589 and the Foundation for Polish Science, START 31.2016 (M. G.). J. T. and D. G. would like to thank Krystian Jażdżewski for helpful discussions.

Conflicts of interest

There are no conflicts to declare.

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Footnotes

Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc05064c
To evaluate the potential of antisense 2′OMeRNA to inhibit translation of the RFP protein, we cultured bacteria in the presence of 2′OMeRNA conjugated to vitamin B12. Escherichia coli K-12 MG165525 and Salmonella enterica subsp. enterica serovar Typhimurium LT2-R20 cells encoding the mrfp1 gene were grown for 20 h in Davis Minimal Broth23 at 37 °C with shaking. The culture was supplemented with kanamycin to prevent plasmid loss. The effect of vitamin B12–2′OMeRNA conjugates on RFP production was determined using a standard microdilution method at a concentration range of 0–16 μM. To obtain the relative fluorescence values (RFU), the previously described method was used.20

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