Several oligothymidylates containing various ratios of phosphodiester and isopolar 5'-hydroxyphosphonate, 5'-O-methylphosphonate and 3'-O-methylphosphonate internucleotide linkages were examined with respect to their hybridization properties with oligoriboadenylates and their ability to induce RNA cleavage by ribonuclease H (RNase H). The results demonstrated that the increasing number of 5'-hydroxyphosphonate or 5'-O-methylphosphonate units in antisense oligonucleotides (AOs) significantly stabilizes the heteroduplexes, whereas 3'-O-methylphosphonate AOs cause strong destabilization of the heteroduplexes. Only the heteroduplexes with 5'-O-methylphosphonate units in the antisense strand exhibited a significant increase in Escherichia coli RNase H cleavage activity by up to 3-fold (depending on the ratio of phosphodiester and phosphonate linkages) in comparison with the natural heteroduplex. A similar increase in RNase H cleavage activity was also observed for heteroduplexes composed of miRNA191 and complementary AOs containing 5'-O-methylphosphonate units. We propose for this type of AOs, working via the RNase H mechanism, the abbreviation MEPNA (MEthylPhosphonate Nucleic Acid).

Institute of Photonics and Electronics AS CR v v i Chaberská 57 182 51 Prague Czech Republic Institute of Organic Chemistry and Biochemistry AS CR v v i Flemingovo nám 2 166 10 Prague Czech Republic and Faculty of Mathematics and Physics Charles University Prague Ke Karlovu 3 121 16 Prague Czech Republic

See more in PubMed

Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer. Oncogene. 2003;22:9087–9096. PubMed

Marwick C. First “antisense” drug will treat CMV retinitis. JAMA. 1998;280:871. PubMed

Crooke ST, Geary RS. Clinical pharmacological properties of mipomersen (Kynamro), a second generation antisense inhibitor of apolipoprotein B. Br. J. Clin. Pharmacol. 2013;76:269–276. PubMed PMC

Minshull J, Hunt T. The use of single-stranded DNA and RNase H to promote quantitative hybrid arrest of translation of mRNA/DNA hybrids in reticulocyte lysate cell-free translations. Nucleic Acids Res. 1986;14:6433–6451. PubMed PMC

Prakash TP, Siwkowski A, Allerson CR, Migawa MT, Lee S, Gaus HJ, Black C, Seth PP, Swayze EE, Bhat B. Antisense oligonucleotides containing conformationally constrained 2′,4′-(N-methoxy)aminomethylene and 2′,4′-aminooxymethylene and 2′-O,4′-C-aminomethylene bridged nucleoside analogues show improved potency in animal models. J. Med. Chem. 2010;53:1636–1650. PubMed

Wheeler TM, Leger AJ, Pandey SK, MacLeod AR, Nakamori M, Cheng SH, Wentworth BM, Bennett CF, Thornton CA. Targeting nuclear RNA for in vivo correction of myotonic dystrophy. Nature. 2012;488:111–115. PubMed PMC

Southwell AL, Skotte NH, Bennett CF, Hayden MR. Antisense oligonucleotide therapeutics for inherited neurodegenerative diseases. Trends Mol. Med. 2012;18: 634–643. PubMed

Kole R, Krainer AR, Altman S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nat. Rev. Drug Discov. 2012;11:125–140. PubMed PMC

Zamaratski E, Pradeepkumar PI, Chattopadhyaya J. A critical survey of the structure-function of the antisense oligo/RNA heteroduplex as substrate for RNase H. J. Biochem. Biophys. Methods. 2001;48:189–208. PubMed

Lima WF, Nichols JG, Wu H, Prakash TP, Migawa MT, Wyrzykiewicz TK, Bhat B, Crooke ST. Structural requirements at the catalytic site of the heteroduplex substrate for human RNase H1 catalysis. J. Biol. Chem. 2004;279:36317–36326. PubMed

Crooke ST. Molecular mechanisms of antisense drugs. Biochimica Et Biophysica Acta-Gene Structure and Expression. 1999;1489:31–44. PubMed

Kean JM, Kipp SA, Miller PS, Kulka M, Aurelian L. Inhibition of herpes simplex virus replication by antisense oligo-2′-O-methylribonucleoside methylphosphonates. Biochemistry. 1995;34:14617–14620. PubMed

Crooke ST, Lemonidis KM, Neilson L, Griffey R, Lesnik EA, Monia BP. Kinetic characteristics of Escherichia coli RNase H1—cleavage of various antisense oligonucleotide-RNA duplexes. Biochem. J. 1995;312:599–608. PubMed PMC

Quartin RS, Brakel CL, Wetmur JG. Number and distribution of methylphosphonate linkages in oligodeoxynucleotides affect exo- and endonuclease sensitivity and ability to form RNase H substrates. Nucleic Acids Res. 1989;17:7253–7262. PubMed PMC

Eckstein F. Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them? Antisense Nucleic Acid Drug Dev. 2000;10:117–121. PubMed

Cummins L, Graff D, Beaton G, Marshall WS, Caruthers MH. Biochemical and physicochemical properties of phosphorodithioate DNA. Biochemistry. 1996;35:8734–8741. PubMed

Vaughn JP, Stekler J, Demirdji S, Mills JK, Caruthers MH, Iglehart JD, Marks JR. Inhibition of the erbB-2 tyrosine kinase receptor in breast cancer cells by phosphoromonothioate and phosphorodithioate antisense oligonucleotides. Nucleic Acids Res. 1996;24:4558–4564. PubMed PMC

Sergueev D, Hasan A, Ramaswamy M, Shaw BR. Boranophosphate oligonucleotides: new synthetic approaches. Nucleos Nucleot. 1997;16:1533–1538.

Wang J, Verbeure B, Luyten I, Froeyen M, Hendrix C, Rosemeyer H, Seela F, Van Aerschot A, Herdewijn P. Cyclohexene nucleic acids (CeNA) form stable duplexes with RNA and induce RNase H activity. Nucleosides Nucleotides Nucleic Acids. 2001;20:785–788. PubMed

Verbeure B, Lescrinier E, Wang J, Herdewijn P. RNase H mediated cleavage of RNA by cyclohexene nucleic acid (CeNA) Nucleic Acids Res. 2001;29:4941–4947. PubMed PMC

Damha MJ, Wilds CJ, Noronha A, Brukner I, Borkow G, Arion D, Parniak MA. Hybrids of RNA and arabinonucleic acids (ANA and 2′ F-ANA) are substrates of ribonuclease H. J. Am. Chem. Soc. 1998;120:12976–12977.

Rait VK, Shaw BR. Boranophosphates support the RNase H cleavage of polyribonucleotides. Antisense Nucleic Acid Drug Dev. 1999;9:53–60. PubMed

Anzahaee MY, Watts JK, Alla NR, Nicholson AW, Damha MJ. Energetically important C-H center dot center dot center dot F-C pseudohydrogen bonding in water: evidence and application to rational design of oligonucleotides with high binding affinity. J. Am. Chem. Soc. 2011;133:728–731. PubMed

Kalota A, Karabon L, Swider CR, Viazovkina E, Elzagheid M, Damha MJ, Gewirtz AM. 2′-deoxy-2′-fluoro-beta-D-arabinonucleic acid (2′F-ANA) modified oligonucleotides (ON) effect highly efficient, and persistent, gene silencing. Nucleic Acids Res. 2006;34:451–461. PubMed PMC

Souleimanian N, Deleavey GF, Soifer H, Wang S, Tiemann K, Damha MJ, Stein CA. Antisense 2′-Deoxy, 2′-fluoroarabino nucleic acid (2′F-ANA) oligonucleotides: in vitro gymnotic silencers of gene expression whose potency is enhanced by fatty acids. Mol. Ther. Nucleic Acids. 2012;1:e43. PubMed PMC

Fortin M, D'Anjou H, Higgins ME, Gougeon J, Aubé P, Moktefi K, Mouissi S, Séguin S, Séguin R, Renzi PM, et al. A multi-target antisense approach against PDE4 and PDE7 reduces smoke-induced lung inflammation in mice. Resp. Res. 2009;10:39. PubMed PMC

Dellinger DJ, Sheehan DM, Christensen NK, Lindberg JG, Caruthers MH. Solid-phase chemical synthesis of phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides. J. Am. Chem. Soc. 2003;125:940–950. PubMed

Sheehan D, Lunstad B, Yamada CM, Stell BG, Caruthers MH, Dellinger DJ. Biochemical properties of phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides. Nucleic Acids Res. 2003;31:4109–4118. PubMed PMC

Rejman D, Masojídková M, Rosenberg I. Nucleosidyl-O-methylphosphonates: a pool of monomers for modified oligonucleotides. Nucleosides Nucleotides Nucleic Acids. 2004;23: 1683–1705. PubMed

Králíková S, Buděšínský M, Barvík I, Masojídková M, Točík Z, Rosenberg I. Synthesis and properties of ApA analogues with shortened phosphonate internucleotide linkage. Nucleosides Nucleotides Nucleic Acids. 2011;30:524–543. PubMed

Šípová H, Vaisocherová H, Štěpánek J, Homola J. A dual surface plasmon resonance assay for the determination of ribonuclease H activity. Biosens Bioelectron. 2010;26:1605–1611. PubMed

Točík Z, Barvík I, Jr, Buděšínský M, Rosenberg I. Novel isosteric, isopolar phosphonate analogs of oligonucleotides: preparation and properties. Biopolymers. 2006;83: 400–413. PubMed

Šípová H, Ševců V, Kuchař M, Ahmad JN, Mikulecký P, Osička R, Malý P, Homola J. Surface plasmon resonance biosensor based on engineered proteins for direct detection of interferon-gamma in diluted blood plasma. Sensor Actuat B Chem. 2012;174:306–311.

Su X, Wu YJ, Robelek R, Knoll W. Surface plasmon resonance spectroscopy and quartz crystal microbalance study of streptavidin film structure effects on biotinylated DNA assembly and target DNA hybridization. Langmuir. 2005;21: 348–353. PubMed

Barvík I. E.coli RNase HI and the phosphonate-DNA/RNA hybrid: molecular dynamics simulations. Nucleosides Nucleotides Nucleic Acids. 2005;24:435–441. PubMed

Nowotny M, Gaidamakov SA, Ghirlando R, Cerritelli SM, Crouch RJ, Yang W. Structure of human RNase H1 complexed with an RNA/DNA hybrid: Insight into HIV reverse transcription. Mol. Cell. 2007;28: 264–276. PubMed

Maláč K, Barvík I. Complex between human RNase HI and the phosphonate-DNA/RNA duplex: molecular dynamics study. J. Mol. Graph Model. 2013;44:81–90. PubMed

Páv O, Košiová I, Barvík I, Pohl R, Buděšínský M, Rosenberg I. Synthesis of oligoribonucleotides with phosphonate-modified linkages. Org. Biomol. Chem. 2011;9: 6120–6126. PubMed

Liboska R, Snášel J, Barvík I, Buděšínský M, Pohl R, Točík Z, Páv O, Rejman D, Novák P, Rosenberg I. 4 ‘-Alkoxy oligodeoxynucleotides: a novel class of RNA mimics. Org. Biomol. Chem. 2011;9:8261–8267. PubMed

Lima WF, Crooke ST. Binding affinity and specificity of Escherichia coli RNase H1: Impact on the kinetics of catalysis of antisense oligonucleotide-RNA hybrids. Biochemistry. 1997;36:390–398. PubMed

Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc. Natl Acad. Sci. USA. 2006;103:2257–2261. PubMed PMC

Lima WF, Rose JB, Nichols JG, Wu HJ, Migawa MT, Wyrzykiewicz TK, Siwkowski AM, Crooke ST. Human RNase H1 discriminates between subtle variations in the structure of the heteroduplex substrate. Mol. Pharmacol. 2007;71:83–91. PubMed

Králiková A, Buděšínský M, Masojídkova M, Rosenberg I. Nucleoside 5′-C-phosphonates: reactivity of the alpha-hydroxyphosphonate moiety. Tetrahedron. 2006;62:4917–4932.

Cerritelli SM, Crouch RJ. Ribonuclease H: the enzymes in eukaryotes. FEBS J. 2009;276:1494–1505. PubMed PMC

Østergaard ME, Southwell AL, Kordasiewicz H, Watt AT, Skotte NH, Doty CN, Vaid K, Villanueva EB, Swayze EE, Bennett CF, et al. Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS. Nucleic Acids Res. 2013;41:9634–9650. PubMed PMC

Solid-Phase Synthesis of Phosphorothioate/Phosphonothioate and Phosphoramidate/Phosphonamidate Oligonucleotides