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. 2018 Jan 19;13(1):e0191138.
doi: 10.1371/journal.pone.0191138. eCollection 2018.

Interactions of 2'-O-methyl oligoribonucleotides with the RNA models of the 30S subunit A-site

Maciej Jasiński  1   2 , Marta Kulik  1 , Monika Wojciechowska  1 , Ryszard Stolarski  3 , Joanna Trylska  1
Affiliations

Interactions of 2'-O-methyl oligoribonucleotides with the RNA models of the 30S subunit A-site

Maciej Jasiński et al. PLoS One. .

Abstract

Synthetic oligonucleotides targeting functional regions of the prokaryotic rRNA could be promising antimicrobial agents. Indeed, such oligonucleotides were proven to inhibit bacterial growth. 2'-O-methylated (2'-O-Me) oligoribonucleotides with a sequence complementary to the decoding site in 16S rRNA were reported as inhibitors of bacterial translation. However, the binding mode and structures of the formed complexes, as well as the level of selectivity of the oligonucleotides between the prokaryotic and eukaryotic target, were not determined. We have analyzed three 2'-O-Me oligoribonucleotides designed to hybridize with the models of the prokaryotic rRNA containing two neighboring aminoglycoside binding pockets. One pocket is the paromomycin/kanamycin binding site corresponding to the decoding site in the small ribosomal subunit and the other one is the close-by hygromycin B binding site whose dynamics has not been previously reported. Molecular dynamics (MD) simulations, as well as isothermal titration calorimetry, gel electrophoresis and spectroscopic studies have shown that the eukaryotic rRNA model is less conformationally stable (in terms of hydrogen bonds and stacking interactions) than the corresponding prokaryotic one. In MD simulations of the eukaryotic construct, the nucleotide U1498, which plays an important role in correct positioning of mRNA during translation, is flexible and spontaneously flips out into the solvent. In solution studies, the 2'-O-Me oligoribonucleotides did not interact with the double stranded rRNA models but all formed stable complexes with the single-stranded prokaryotic target. 2'-O-Me oligoribonucleotides with one and two mismatches bound less tightly to the eukaryotic target. This shows that at least three mismatches between the 2'-O-Me oligoribonucleotide and eukaryotic rRNA are required to ensure target selectivity. The results also suggest that, in the ribosome environment, the strand invasion is the preferred binding mode of 2'-O-Me oligoribonucleotides targeting the aminoglycoside binding sites in 16S rRNA.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Paromomycin (purple) and hygromycin B (yellow) in their primary binding sites in the rRNA helix h44 of the 30S subunit of the bacterial ribosomes.
RNA is in green and proteins in cyan. Red denotes the rRNA fragment included in the studied model of the prokaryotic rRNA (PDB code: 3LOA [6]). (a) The position of the antibiotics in the 30S subunit. (b) Zoom of paromomycin binding site (PDB code: 2Z4K [7]). (c) Zoom of hygromycin B binding site (PDB code: 3DF3 [8]).
Fig 2
Fig 2. Secondary structures of the eukaryotic and prokaryotic targets and 2’-O-Me oligoribonucleotides.
Numbering of all nucleotides is as in the E. coli ribosome. Nucleotides coloured in the target structures are specific for the H. sapiens and E. coli ribosomes. Nucleotides in the base pair range of 1401–1501 and 1412–1488 are identical as in the small ribosomal subunits of relevant organisms. Four additional base pairs at each terminus of the model were added, the same as in the work of Dibrov et al. [6] to stabilize the termini.
Fig 3
Fig 3. Solution studies of rRNA models.
(a) 2AP fluorescence intensity during titration of the 2AP-labeled strand B (at the 1493 position) to strand A. (b) UV-melting profiles of the prokaryotic (Tm = 52.9°C) and eukaryotic (Tm = 42.6°C) decoding A-site models presented as the fraction of the double stranded structures versus temperature.
Fig 4
Fig 4. ITC scans of the titration of the A strand to the B strand of the prokaryotic and eukaryotic A-site models at T = 21°C.
Fig 5
Fig 5. Trajectory derived RMSF for the nucleotides of the prokaryotic and eukaryotic models of rRNA A-site, averaged over 900 ns. For sequences see Fig 2.
Fig 6
Fig 6. Properties derived from the trajectories of the eukaryotic and prokaryotic RNA models of the decoding A-site.
(a) RMSD calculated for RNA heavy atoms with respect to the starting structure, (b) the sum of electrostatic and VdW interactions between the stacking nucleobases, (c) the number of hydrogen bonds in the WC-WC pairs, (d) the number of hydrogen bonds in the non-WC-WC pairs. Each point represents a 5 ns average of each property.
Fig 7
Fig 7. Representative structures of the (a) eukaryotic and (b) prokaryotic RNA models from MD simulations.
Central structures from the most occupied clusters with the RMSD criterion of 2 Å. Nucleotides are coloured according to the NDB database convention: A red, U cyan, C yellow, G green [66]. Fragments identical as in the small ribosomal subunits of the relevant organisms a) H. sapiens, b) E. coli are highlighted. Black ribbon represents the strand complementary to the 2’-O-Me oligonucleotides.
Fig 8
Fig 8. Average secondary structures of the: (a–d) prokaryotic and (e–h) eukaryotic system from the last 900 ns of MD simulations.
Only WC-WC pairs that were present in more than 50% of the simulation time are shown. Nucleotides are coloured by the: (a, e) average number of hydrogen bonds in the WC-WC pairs, (b, f) average number of hydrogen bonds in the non-WC-WC pairs, (c, g) average Coulomb energy [kcal/mol], (d, h) average VdW energy [kcal/mol].
Fig 9
Fig 9. Conformational mobility of A1492, A1493 and U1498.
(a) Plots of pseudo-dihedral angles representing the orientation of the nucleobase with respect to the helix backbone for the eukaryotic and prokaryotic model. For angle definition see Methods. (b) Trajectory snapshots presenting the flipping events in the eukaryotic model in comparison with the representative structure of the first most populated cluster from the simulation of the prokaryotic model.
Fig 10
Fig 10. Solution studies of the interactions between the 2’-O-Me oligomers and rRNA targets.
(a, b) Fluorescence of 2AP1493 in the rRNA strand B monitored during titration of 2’-O-Me oligomers to the rRNA targets. B stands for the single B strand of the rRNA model, AB stands for the double stranded model. (c, d) UV melting profiles of double-stranded models (consisting of rRNA strand B and 2’-O-Me oligomers) showing the fraction of the double strands as a function of temperature (c) and normalized absorbance (d) since fitting was impossible due to the lack of plateaus. For the sequences and names refer to Fig 2.
Fig 11
Fig 11. PAGE studies of the interactions between the 2’-O-Me oligomers and strand B of the (a) prokaryotic and (b) eukaryotic rRNA targets.
For the sequence of strand B and oligomers see Fig 2.
Fig 12
Fig 12. ITC binding scans showing the titration of 2’-O-Me-RNA oligomers to strand B of the prokaryotic rRNA model at T = 21°C.
(a) oligomer 1489, (b) oligomer 1490, (c) oligomer 1491.
Fig 13
Fig 13. The C1402:C4104 and G1497:A1500 region of hygromycin B binding site in the crystal and cryo-electron microscopy structures of the prokaryotic small ribosomal subunit.
U1498 is in light green, hygromycin B in pink and mRNA in violet. PDB codes are given next to each structure. Prokaryotic and eukaryotic “1st cluster” refers to representative structures of the most occupied clusters obtained in MD simulations with the RMSD criterion of 2 Å.
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Grants and funding

J. T and M. W. thank the National Science Centre (grant no DEC-2014/12/W/ST5/00589 in Symfonia call) and CeNT BST funds. M. J. thanks the European Social Fund (contract no UDA-POKL.04.01.01-00-072/09-00). Simulations were performed at the Interdisciplinary Centre for Mathematical and Computational Modelling (grants G31-4 and GA65-17) and Centre of New Technologies, University of Warsaw. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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