Single-stranded (ss) 2′-fluoro (2′-F)-modified oligonucleotides (ONs) with a full phosphorothioate (PS) backbone have been reported to be cytotoxic and cause DNA double-strand breaks (DSBs) when transfected into HeLa cells. However, the molecular determinants of these effects have not been fully explored. In this study, we investigated the impact of ON structure, chemistry, delivery method, and cell type on in vitro cytotoxicity and DSBs. We found that ss PS-ONs were more cytotoxic than double-stranded (ds) PS-ONs, irrespective of the 2′-ribose chemistry, inclusive of the 2′-F modification. Cytotoxicity of ss ONs was most affected by the total PS content, with an additional contribution of 2′-F substitutions in HeLa, but not HepG2, cells. The relatively mild cytotoxicity of ds ONs was most impacted by long contiguous PS stretches combined with 2′-F substitutions. None of the tested ds 2′-F-modified PS-ONs caused DSBs, while the previously reported DSBs caused by ss 2′-F-modified PS-ONs were PS dependent. HeLa cells were more sensitive to ON-mediated toxicity when transfected with Lipofectamine 2000 versus Lipofectamine RNAiMax. Importantly, asialoglycoprotein receptor-mediated uptake of N-acetylgalactosamine-conjugated ss or ds PS-ONs, even those with long PS stretches and high 2′-F content, was neither cytotoxic nor caused DSBs at transfection-equivalent exposures. These results suggest that in vitro cytotoxicity and DSBs associated with ONs are delivery method dependent and primarily determined by single-stranded nature and PS content of ONs.
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References
1.
LundinKE, GissbergO and SmithC.I.. (2015) Oligonucleotide therapies: the past and the present. Hum Gene Ther, 26:475–485.
2.
WittrupA and LiebermanJ. (2015). Knocking down disease: a progress report on siRNA therapeutics. Nat Rev Genet, 16:543–552.
3.
ManoharanM. (2004). RNA interference and chemically modified small interfering RNAs. Curr Opin Chem Biol, 8:570–579.
4.
BumcrotD, ManoharanM, KotelianskyV and SahDW. (2006). RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat Chem Biol, 2:711–719.
5.
EcksteinF. (1985). Nucleoside phosphorothioates. Annu Rev Biochem, 54:367–402.
6.
EcksteinF. (2002). Developments in RNA chemistry, a personal view. Biochimie, 84:841–848.
7.
EcksteinF. (2000). Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them?. Antisense Nucleic Acid Drug Dev, 10:117–121.
8.
BrownDA, KangSH, GryaznovSM, DeDionisioL, HeidenreichO, SullivanS, XuX and NerenbergMI. (1994). Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding. J Biol Chem, 269:26801–26805.
9.
MouTC and GrayDM. (2002). The high binding affinity of phosphorothioate-modified oligomers for Ff gene 5 protein is moderated by the addition of C-5 propyne or 2′-O-methyl modifications. Nucleic Acids Res, 30:749–758.
10.
GearyRS, YuRZ and LevinAA. (2001). Pharmacokinetics of phosphorothioate antisense oligodeoxynucleotides. Curr Opin Investig Drugs, 2:562–573.
11.
WatanabeTA, GearyRS and LevinAA. (2006). Plasma protein binding of an antisense oligonucleotide targeting human ICAM-1 (ISIS 2302). Oligonucleotides, 16:169–180.
12.
BennettCF and SwayzeEE. (2010). RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu Rev Pharmacol Toxicol, 50:259–293.
13.
LorenzP, MisteliT, BakerBF, BennettCF and SpectorDL. (2000). Nucleocytoplasmic shuttling: a novel in vivo property of antisense phosphorothioate oligodeoxynucleotides. Nucleic Acids Res, 28:582–592.
14.
LorenzP, BakerBF, BennettCF and SpectorDL. (1998). Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies. Mol Biol Cell, 9:1007–1023.
15.
SlimG and GaitMJ. (1991). Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. Nucleic Acids Res, 19:1183–1188.
16.
SpitzerS and EcksteinF. (1988). Inhibition of deoxyribonucleases by phosphorothioate groups in oligodeoxyribonucleotides. Nucleic Acids Res, 16:11691–11704.
17.
WhiteAP, ReevesKK, SnyderE, FarrellJ, PowellJW, MohanV and GriffeyRH. (1996). Hydration of single-stranded phosphodiester and phosphorothioate oligodeoxyribonucleotides. Nucleic Acids Res, 24:3261–3266.
18.
KlingJ. (2010). Safety signal dampens reception for mipomersen antisense. Nat Biotechnol, 28:295–297.
19.
HenrySP, BolteH, AulettaC and KornbrustDJ. (1997). Evaluation of the toxicity of ISIS 2302, a phosphorothioate oligonucleotide, in a four-week study in cynomolgus monkeys. Toxicology, 120:145–155.
20.
SteinCA, SubasingheC, ShinozukaK and CohenJS. (1988). Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res, 16:3209–3221.
21.
ClarkCL, CecilPK, SinghD and GrayDM. (1997). CD, absorption and thermodynamic analysis of repeating dinucleotide DNA, RNA and hybrid duplexes [d/r(AC)]12.[d/r(GT/U)]12 and the influence of phosphorothioate substitution. Nucleic Acids Res, 25:4098–4105.
22.
DeleaveyGF and DamhaMJ. (2012). Designing chemically modified oligonucleotides for targeted gene silencing. Chem Biol, 19:937–954.
23.
MoniaBP, LesnikEA, GonzalezC, LimaWF, McGeeD, GuinossoCJ, KawasakiAM, CookPD, and FreierSM. (1993). Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene expression. J Biol Chem, 268:14514–14522.
24.
ManoharanM. (1999). 2'-carbohydrate modifications in antisense oligonucleotide therapy: importance of conformation, configuration and conjugation. Biochim Biophys Acta, 1489:117–130.
25.
PallanPS, GreeneEM, JicmanPA, PandeyRK, ManoharanM, RoznersE and EgliM. (2011). Unexpected origins of the enhanced pairing affinity of 2'-fluoro-modified RNA. Nucleic Acids Res, 39:3482–3495.
26.
WattsJK, DeleaveyGF and DamhaMJ. (2008). Chemically modified siRNA: tools and applications. Drug Discov Today, 13:842–855.
27.
ShenW, LiangXH, SunH and CrookeST. (2015). 2'-Fluoro-modified phosphorothioate oligonucleotide can cause rapid degradation of P54nrb and PSF. Nucleic Acids Res, 43:4569–4578.
28.
WhiteheadKA, DahlmanJE, LangerRS and AndersonDG. (2011). Silencing or stimulation? siRNA delivery and the immune system. Annu Rev Chem Biomol Eng, 2:77–96.
29.
RettigGR and BehlkeMA. (2012). Progress toward in vivo use of siRNAs-II. Mol Ther, 20:483–512.
30.
WahlestedtC, SalmiP, GoodL, KelaJ, JohnssonT, HokfeltT, BrobergerC, PorrecaF, LaiJ, RenK, et al. (2000). Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc Natl Acad Sci U S A, 97:5633–5638.
31.
Ten AsbroekAL, van GroenigenM, NooijM and BaasF. (2002). The involvement of human ribonucleases H1 and H2 in the variation of response of cells to antisense phosphorothioate oligonucleotides. Eur J Biochem, 269:583–592.
32.
WardAJ, NorrbomM, ChunS, BennettCF and RigoF. (2014). Nonsense-mediated decay as a terminating mechanism for antisense oligonucleotides. Nucleic Acids Res, 42:5871–5879.
33.
VickersTA, FreierSM, BuiHH, WattA and Crooke.ST (2014). Targeting of repeated sequences unique to a gene results in significant increases in antisense oligonucleotide potency. PLoS One, 9:e110615.
34.
LeungAK, CalabreseJM and SharpPA. (2006). Quantitative analysis of Argonaute protein reveals microRNA-dependent localization to stress granules. Proc Natl Acad Sci U S A, 103:18125–18130.
35.
BermanCL, BarrosSA, GallowaySM, KasperP, OlesonFB, PriestleyCC, SwederKS, SchlosserMJ and SobolZ. (2016). OSWG recommendations for genotoxicity testing of novel oligonucleotide-based therapeutics. Nucleic Acid Ther, 26:73–85.
36.
JanasMM, JiangY, DuncanRG, HayesAN, LiuJ, KasperkovitzPV, PlackeME and BarrosSA. (2016). Exposure to siRNA-GalNAc conjugates in systems of the standard test battery for genotoxicity. Nucleic Acid Ther, 26:363–371.
37.
NairJK, WilloughbyJL, ChanA, CharisseK, AlamMR, WangQ, HoekstraM, KandasamyP, Kel'inAV, MilsteinS, et al. (2014). Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J Am Chem Soc, 136:16958–16961.
38.
BurelSA, HanSR, LeeHS, NorrisDA, LeeBS, MachemerT, ParkSY, ZhouT, HeG, KimY, et al. (2013). Preclinical evaluation of the toxicological effects of a novel constrained ethyl modified antisense compound targeting signal transducer and activator of transcription 3 in mice and cynomolgus monkeys. Nucleic Acid Ther, 23:213–227.
39.
PrakashTP, GrahamMJ, YuJ, CartyR, LowA, ChappellA, SchmidtK, ZhaoC, AghajanM, MurrayHF, et al. (2014). Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res, 42:8796–8807.
40.
WitzigmannD, QuagliataL, SchenkSH, QuintavalleC, TerraccianoLM and HuwylerJ. (2015). Variable asialoglycoprotein-receptor 1 expression in liver disease: implications for therapeutic intervention. Hepatol Res, 46:686–696.
41.
CapodiciJ, KarikoK and WeissmanD. (2002). Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference. J Immunol, 169:5196–5201.
42.
ZhouJ and RossiJJ. (2011). Cell-specific aptamer-mediated targeted drug delivery. Oligonucleotides, 21:1–10.
43.
LayzerJM, McCaffreyAP, TannerAK, HuangZ, KayMA and SullengerBA. (2004). In vivo activity of nuclease-resistant siRNAs. RNA, 10:766–771.
44.
ChiuYL and RanaTM. (2003). siRNA function in RNAi: a chemical modification analysis. RNA, 9:1034–1048.
45.
OhrtT and SchwilleP. (2008). siRNA modifications and sub-cellular localization: a question of intracellular transport?. Curr Pharm Des, 14:3674–3685.
46.
MarcussonEG, BhatB, ManoharanM, BennettCF and DeanNM. (1998). Phosphorothioate oligodeoxyribonucleotides dissociate from cationic lipids before entering the nucleus. Nucleic Acids Res, 26:2016–2023.
47.
ZelphatiO and SzokaFCJr., (1996). Mechanism of oligonucleotide release from cationic liposomes. Proc Natl Acad Sci U S A, 93:11493–11498.
48.
BennettCF, ChiangMY, ChanH, ShoemakerJE and MirabelliCK. (1992). Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol Pharmacol, 41:1023–1033.
49.
FisherTL, TerhorstT, CaoX and WagnerRW. (1993). Intracellular disposition and metabolism of fluorescently-labeled unmodified and modified oligonucleotides microinjected into mammalian cells. Nucleic Acids Res, 21:3857–3865.
50.
PichonC, FreulonI, MidouxP, MayerR, MonsignyM and RocheAC. (1997). Cytosolic and nuclear delivery of oligonucleotides mediated by an amphiphilic anionic peptide. Antisense Nucleic Acid Drug Dev, 7:335–343.
51.
CastanottoD, LinM, KowolikC, WangL, RenXQ, SoiferHS, KochT, HansenBR, OerumH, ArmstrongB, et al. (2015). A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells. Nucleic Acids Res, 43:9350–9361.
52.
ZhaoM, YangH, JiangX, ZhouW, ZhuB, ZengY, YaoK and RenC. (2008). Lipofectamine RNAiMAX: an efficient siRNA transfection reagent in human embryonic stem cells. Mol Biotechnol, 40:19–26.
53.
LiangXH, ShenW, SunH, PrakashTP and CrookeST. (2014). TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells. Nucleic Acids Res, 42:7819–7832.
54.
JonesS, DaleyDT, LuscombeNM, BermanHM and ThorntonJM. (2001). Protein-RNA interactions: a structural analysis. Nucleic Acids Res, 29:943–954.
55.
ShenW, LiangXH and CrookeST. (2014). Phosphorothioate oligonucleotides can displace NEAT1 RNA and form nuclear paraspeckle-like structures. Nucleic Acids Res, 42:8648–8662.
56.
PendergraffHM, DebackerAJ and WattsJK. (2016). Single-stranded silencing RNAs: hit rate and chemical modification. Nucleic Acid Ther, 26:216–222.
57.
TeplovaM, MinasovG, TereshkoV, InamatiGB, CookPD, ManoharanM and EgliM. (1999). Crystal structure and improved antisense properties of 2'-O-(2-methoxyethyl)-RNA. Nat Struct Biol, 6:535–539.
58.
EgliM, PortmannS and UsmanN. (1996). RNA hydration: a detailed look. Biochemistry, 35:8489–8494.
59.
HarborthJ, ElbashirSM, VandenburghK, ManningaH, ScaringeSA, Weber K and TuschlT. (2003). Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev, 13:83–105.
60.
SchwartzAL. (1991). Trafficking of asialoglycoproteins and the asialoglycoprotein receptor. Targeted Diagn Ther, 4:3–39.
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