Scanning of 16S Ribosomal RNA for Peptide Nucleic Acid Targets

Górska, Anna; Markowska-Zagrajek, Agnieszka; Równicki, Marcin; Trylska, Joanna

doi:10.1021/acs.jpcb.6b02081

Cited by 17 publications

(16 citation statements)

References 44 publications

(72 reference statements)

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“…The solvent accessible surface area of this 516–528 stretch was calculated as 1921 Å 2 so it was assumed accessible. The energetic cost of opening of this fragment was estimated as −0.9 ± 0.8 kcal mol −1 , suggesting that strand‐invasion by oligomers is possible . We thus anticipated that oligonucleotides complementary to the 516–528 loop would hybridize with helix 18.…”

Section: Resultsmentioning

confidence: 99%

“…Single base substitutions that disrupted the Watson‐Crick pairs in the helix18 pseudo‐knot were often deleterious in E. coli and caused resistance to streptomycin . The alignment of helix 18 sequences of the human and E. coli rRNA shows low homology (22 nucleotide differences) . These sequence differences between the E. coli and human helix 18, combined with its accessibility and functional significance, could make helix 18 a possible target for species selective ribosome inhibition …”

Section: Introductionmentioning

confidence: 99%

“…[7] The alignment of helix 18 sequences of the human and E. coli rRNA shows low homology (22 nucleotide differences). [22] These sequence differences between the E. coli and human helix 18, combined with its accessibility and functional significance, could make helix 18 a possible target for species selective ribosome inhibition. [21] In this work we investigated the structure and thermodynamics of helix 18, specifically the formation of the pseudo-knot, in the absence of the ribosome context.…”

Section: Introductionmentioning

confidence: 99%

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Thermodynamics of the pseudo‐knot in helix 18 of 16S ribosomal RNA

2018

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A fragment of E. coli 16S rRNA formed by nucleotides 500 to 545 is termed helix 18. Nucleotides 505-507 and 524-526 form a pseudo-knot and its distortion affects ribosome function. Helix 18 isolated from the ribosome context is thus an interesting fragment to investigate the structural properties and folding of RNA with pseudo-knots. With all-atom molecular dynamics simulations, spectroscopic and gel electrophoresis experiments, we investigated thermodynamics of helix 18, with a focus on its pseudo-knot. In solution studies at ambient conditions we observed dimerization of helix 18. We proposed that the loop, containing nucleotides forming the pseudo-knot, interacts with another monomer of helix 18. The native dimer is difficult to break but introducing mutations in the pseudo-knot indeed assured a monomeric form of helix 18. Molecular dynamics simulations at 310 K confirmed the stability of the pseudo-knot but at elevated temperatures this pseudo-knot was the first part of helix 18 to lose the hydrogen bond pattern. To further determine helix 18 stability, we analyzed the interactions of helix 18 with short oligomers complementary to a nucleotide stretch containing the pseudo-knot. The formation of higher-order structures by helix 18 impacts hybridization efficiency of peptide nucleic acid and 2'-O methyl RNA oligomers.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Thermodynamics of the pseudo‐knot in helix 18 of 16S ribosomal RNA

2018

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“…First, the reference free energy of the native rRNA structure of the target (isolated from the ribosome and bare of proteins) was computed from the crystal structure. Second, the cost of opening the rRNA fragments was calculated by subtracting the free energy of the open conformation from the native conformation . The more negative the difference in free energy, the more stable the native (crystallographic) structure of the target, which should correspond to reduced ability of riRNAs to bind to these rRNA fragments.…”

Section: Methodsmentioning

confidence: 99%

Analysis of ribosomal inter‐subunit sites as targets for complementary oligonucleotides

et al. 2017

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The bacterial ribosome has many functional ribosomal RNA (rRNA) sites. We have computationally analyzed the rRNA regions involved in the interactions between the 30S and 50S subunits. Various properties of rRNA such as solvent accessibility, opening energy, hydrogen bonding pattern, van der Waals energy, thermodynamic stability were determined. Based on these properties we selected rRNA targets for hybridization with complementary 2'-O-methyl oligoribonucleotides (2'-OMe RNAs). Further, the inhibition efficiencies of the designed ribosome-interfering 2'-OMe RNAs were tested using a β-galactosidase assay in a translation system based on the E. coli extract. Several of the oligonucleotides displayed IC values below 1 μM, which were in a similar range as those determined for known ribosome inhibitors, tetracycline and pactamycin. The calculated opening and van der Waals stacking energies of the rRNA targets correlated best with the inhibitory efficiencies of 2'-OMe RNAs. Moreover, the binding affinities of several oligonucleotides to both 70S ribosomes and isolated 30S and 50S subunits were measured using a double-filter retention assay. Further, we applied heat-shock chemical transformation to introduce 2'-OMe RNAs to E. coli cells and verify inhibition of bacterial growth. We observed high correlation between IC in the cell-free extract and bacterial growth inhibition. Overall, the results suggest that the computational analysis of potential rRNA targets within the conformationally dynamic regions of inter-subunit bridges can help design efficient antisense oligomers to probe the ribosome function.

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“…This approach would help in improving patient's health by fast and appropriate antimicrobial intervention (Nölling et al, 2016). Gorska et al (Górska et al, 2016) scanned various target sites in 16S (Haaima et al, 1997) 2. Pseudoiso-cytosine Mimics the C + recognition pattern for triplex formation irrespective of surrounding pH (Haaima et al, 1997) 3.…”

Section: Antibacterial Agents and Antibioticsmentioning

confidence: 99%

Peptide nucleic acids: Advanced tools for biomedical applications

Gupta

Mishra

Puri³

2017

Journal of Biotechnology

143

120

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Peptide Nucleic Acids (PNAs) are the DNA/RNA analogues in which sugar-phosphate backbone is replaced by N-2-aminoethylglycine repeating units. PNA contains neutral backbone hence due to the absence of electrostatic repulsion, its hybridization shows remarkable stability towards complementary oligonucleotides. PNAs are highly resistant to cleavage by chemicals and enzymes due to the substrate specific nature of enzymes and therefore not degraded inside the cells. PNAs are emerging as new tools in the market due to their applications in antisense and antigene therapies by inhibiting translation and transcription respectively. Hence, several methods based on PNAs have been developed for designing various anticancer and antigene drugs, detection of mutations or modulation of PCR reactions. The duplex homopurine sequence of DNA may also be recognized by PNA, forming firm PNA/DNA/PNA triplex through strand invasion with a looped-out DNA strand. PNAs have also been found to replace DNA probes in varied investigative purposes. There are several disadvantages regarding cellular uptake of PNA, so modifications in PNA backbone or covalent coupling with cell penetrating peptides is necessary to improve its delivery inside the cells. In this review, hybridization properties along with potential applications of PNA in the field of diagnostics and pharmaceuticals are elaborated.

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Scanning of 16S Ribosomal RNA for Peptide Nucleic Acid Targets

Cited by 17 publications

References 44 publications

Thermodynamics of the pseudo‐knot in helix 18 of 16S ribosomal RNA

Thermodynamics of the pseudo‐knot in helix 18 of 16S ribosomal RNA

Analysis of ribosomal inter‐subunit sites as targets for complementary oligonucleotides

Peptide nucleic acids: Advanced tools for biomedical applications

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