Molecular Biology

, Volume 52, Issue 6, pp 905–912 | Cite as

Modified Oligonucleotides for Guiding RNA Cleavage Using Bacterial RNase P

  • D. S. NovopashinaEmail author
  • A. S. Nazarov
  • M. A. Vorobjeva
  • M. S. Kuprushkin
  • A. S. Davydova
  • A. A. Lomzov
  • D. V. Pyshnyi
  • S. Altman
  • A. G. Venyaminova


The ability of a series of novel modified external guide sequences (EGS oligonucleotides) to induce the hydrolysis of target RNA with bacterial ribonuclease P has been studied; the most efficient modification variants have been selected. We have found patterns of the oligonucleotide sugar-phosphate backbone modi-fications that enhance oligonucleotide stability in the biological environment and do not violate the ability to interact with the enzyme and induce the RNA hydrolysis. It has been shown that analogues of EGS oligonucleotides selectively modified at 2'-position (2'-O-methyl and 2'-fluoro) or at internucleotide phosphates (phosphoryl guanidines) can be used for the addressed cleavage of a model RNA target by bacterial RNase P. The ability of new phosphoryl guanidine analogues of oligodeoxyribonucleotides that are stable in biological media to induce the hydrolysis of target RNA with bacterial ribonuclease P has been shown for the first time. The modified EGS oligonucleotides with an optimal balance between functional activity and stability in biological media can be considered as potential antibacterial agents.


bacterial RNase P EGS oligonucleotides modified oligonucleotides oligo(2'-О-methylribonucleotides) 2'-fluoro modified oligoribonucleotides phosphoryl guanidine oligonucleotides 



  1. 1.
    Llor C., Bjerrum L. 2014. Antimicrobial resistance: Risk associated with antibiotic overuse and initiatives to reduce the problem. Ther. Adv. Drug Saf. 5, 229–241.CrossRefGoogle Scholar
  2. 2.
    Guidry C.A., Mansfield S.A., Cook C.H., Sawyer R.G. 2014. Resistant pathogens, fungi, and viruses. Surg. Clin. North Am. 94, 1195–1218.CrossRefGoogle Scholar
  3. 3.
    Bai H., Xue X., Hou Z., Zhou Y., Meng J., Luo X. 2010. Antisense antibiotics: A brief review of novel target discovery and delivery. Curr. Drug Discov. Technol. 7, 76–85.CrossRefGoogle Scholar
  4. 4.
    Walker S.C., Engelke D.R. 2006. Ribonuclease P: The evolution of an ancient RNA enzyme. Crit. Rev. Biochem. Mol. Biol. 41, 77–102.CrossRefGoogle Scholar
  5. 5.
    Forster A.C., Altman S. 1990. External guide sequences for an RNA molecule. Science. 249, 783–786.CrossRefGoogle Scholar
  6. 6.
    Derksen M., Mertens V., Pruijn G.J. 2015. RNase P-mediated sequence-specific cleavage of RNA by engineered external guide sequences. Biomolecules. 5, 3029–3050.CrossRefGoogle Scholar
  7. 7.
    Lundblad E. W., Altman S. 2010. Inhibition of gene expression by RNase P. Nat. Biotechnol. 27, 212–221.Google Scholar
  8. 8.
    Wesolowski D., Tae H.S., Gandotra N., Llopis P., Shen N., Altman S. 2011. Basic peptide-morpholino oligomer conjugate that is very effective in killing bacteria by gene-specific and nonspecific modes. Proc. Natl. Acad. Sci. U. S. A. 108, 16582‒16587.CrossRefGoogle Scholar
  9. 9.
    Wesolowski D., Alonso D., Altman S. 2013. Combined effect of a peptide–morpholino oligonucleotide conjugate and a cell-penetrating peptide as an antibiotic. Proc. Natl. Acad. Sci. U. S. A. 110, 8686–8689.CrossRefGoogle Scholar
  10. 10.
    Soler Bistué A.J.C., Martín F.A., Vozza N., Ha H., Joaquín J.C., Zorreguieta A., Tolmasky M.E. 2009. Inhibition of AAC(6')-Ib-mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria. Proc. Natl. Acad. Sci. U. S. A. 106, 13230–13235.CrossRefGoogle Scholar
  11. 11.
    Jackson A., Jani S., Sala C.D., Soler-Bistué A.J., Zorreguieta A., Tolmasky M.E. 2016. Assessment of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: A metho-dology for inhibition of expression of antibiotic resistance genes. Biol. Methods Protoc. 1, bpw001.Google Scholar
  12. 12.
    Shen N., Ko J., Xiao G., Wesolowski D., Shan G., Geller B., Izadjoo M., Altman S. 2009. Inactivation of expression of several genes in a variety of bacterial species by EGS technology. Proc. Natl. Acad. Sci. U. S. A. 106, 8163–8168.CrossRefGoogle Scholar
  13. 13.
    Augagneur Y., Wesolowski D., Tae H.S., Altman S., Ben Mamoun C. 2012. Gene selective mRNA cleavage inhibits the development of Plasmodium falciparum. Proc. Natl. Acad. Sci. U. S. A. 109, 6235–6240.CrossRefGoogle Scholar
  14. 14.
    Sala C.D., Soler-Bistué A.J.C., Korprapun L., Zorreguieta A., Tolmasky M.E. 2012. Inhibition of cell division induced by external guide sequences (EGS Technology) targeting ftsZ. PLoS One. 7, 1–7.Google Scholar
  15. 15.
    Sawyer A.J., Wesolowski D., Gandotra N., Stojadinovic A., Izadjoo M., Altman S., Kyriakides T.R. 2013. A peptide–morpholino oligomer conjugate targeting Staphylococcus aureus gyrA mRNA improves healing in an infected mouse cutaneous wound model. Int. J. Pharm. 453, 651–655.CrossRefGoogle Scholar
  16. 16.
    Guerrier-Takada C., Gardiner K., Marsh T., Pace N., Altman S. 1983. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell. 35, 849–857.CrossRefGoogle Scholar
  17. 17.
    Guerrier-Takada C., Lumelsky N., Altman S. 1989. Specific interactions in RNA enzyme–substrate complexes. Science. 246, 1578–1584.CrossRefGoogle Scholar
  18. 18.
    Kupryushkin M.S., Pyshnyi D.V., Stetsenko D.A. 2014. Phosphoryl guanidines: A new class of nucleic acid analogs. Acta Naturae. 6, 123–125.Google Scholar
  19. 19.
    Stetsenko D.A., Kupryushkin M.S., Pyshnyi D.V. 2014. Modified oligonucleotides and methods for their synthesis. Patent WO2016028187 A1. Priority date August 22, 2014.Google Scholar
  20. 20.
    Jiang X., Sunkara N., Lu S., Liu F. 2014. Directing RNase P-mediated cleavage of target mRNAs by engineered external guide sequences in cultured cells. Ther. Appl. Ribozymes Riboswitches. 1103, 45–56.CrossRefGoogle Scholar
  21. 21.
    Sawa T. 2014. The molecular mechanism of acute lung injury caused by Pseudomonas aeruginosa: From bacterial pathogenesis to host response. J. Intensive Care. 2, 10. doi 10.1186/2052-0492-2-10CrossRefGoogle Scholar
  22. 22.
    Perreault J.P., Altman S. 1992. Important 2'-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli. J. Mol. Biol. 226, 399–409.CrossRefGoogle Scholar
  23. 23.
    Sabatino D., Damha M.J. 2007. Oxepane nucleic acids: Synthesis, characterization, and properties of oligonucleotides bearing a seven-membered carbohydrate ring. J. Am. Chem. Soc. 129, 8259–8270.CrossRefGoogle Scholar
  24. 24.
    Ge Q., Dallas A., Ilves H., Shorenstein J., Behlke M.A., Johnston B.H. 2010. Effects of chemical modification on the potency, serum stability, and immunostimulatory properties of short shRNAs. RNA. 16, 118–130.CrossRefGoogle Scholar
  25. 25.
    Li P., Sun J., Su M., Yang X., Tang X. 2014. Design, synthesis and properties of artificial nucleic acids from (R)-4-amino-butane-1,3-diol. Org. Biomol. Chem. 12, 2263–2272.CrossRefGoogle Scholar
  26. 26.
    Soler Bistué A.J., Ha H., Sarno R., Don M., Zorreguieta A., Tolmasky M.E. 2007. External guide sequences targeting the aac(6')-Ib mRNA induce inhibition of amikacin resistance. Antimicrob. Agents Chemother. 51, 1918–1925.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2018

Authors and Affiliations

  • D. S. Novopashina
    • 1
    • 2
    Email author
  • A. S. Nazarov
    • 1
    • 2
  • M. A. Vorobjeva
    • 1
  • M. S. Kuprushkin
    • 1
  • A. S. Davydova
    • 1
  • A. A. Lomzov
    • 1
    • 2
  • D. V. Pyshnyi
    • 1
    • 2
  • S. Altman
    • 3
    • 4
  • A. G. Venyaminova
    • 1
  1. 1.Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of SciencesNovosibirskRussia
  2. 2.Novosibirsk State UniversityNovosibirskRussia
  3. 3.Department of Molecular, Cellular and Developmental Biology, Yale UniversityNew Haven,USA
  4. 4.Division of Life Sciences, Arizona State UniversityTempe, AZ, USA

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