A series of siRNA duplexes containing cationic non-bridging 3′,5′-linked phosphoramidate (PN) linkages was designed and synthesized using a combination of phosphoramidite and H-phosphonate chemistries. Modified oligonucleotides were assayed for their thermal stability, helical structure, and ability to modulate the expression of firefly luciferase. We demonstrate that PN modifications of siRNAs are, in general, minimally destabilizing with respect to duplex thermal stability; destabilization can be mitigated through the incorporation of 2′-modified RNA-like residues or PN conjugates containing ionizable pendant moieties. We also demonstrate that single cationic dimethylethylenediamine PN linkages have little effect on siRNA potency, whether located in the passenger or guide strand of the duplex. Highly modified siRNA passenger strands were further modified with up to four cationic PN linkages, with little effect on duplex potency or helical structure. We envision that PN modifications could be useful in the production of therapeutic siRNAs with optimal biological properties.
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References
1.
ElbashirSM, HarborthJ, LendeckelW, YalcinA, WeberK and TuschlT. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411:494–498.
2.
ManoharanM. (2004). RNA interference and chemically modified small interfering RNAs. Curr Opin Chem Biol, 8:570–579.
3.
BumcrotD, ManoharanM, KotelianskyV and SahDWY. (2006). RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat Chem Biol, 2:711–719.
4.
DeleaveyGF and DamhaMJ. (2012). Designing chemically modified oligonucleotides for targeted gene silencing. Chem Biol, 19:937–954.
5.
SharmaVK and WattsJK. (2015). Oligonucleotide therapeutics: chemistry, delivery and clinical progress. Future Med Chem, 7:2221–2242.
MutisyaD, HardcastleT, CheruiyotSK, PallanPS, KennedySD, EgliM, KelleyML, SmithAVB and RoznersE. (2017). Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs. Nucleic Acids Res, 45: 8142–8155.
10.
EcksteinF. (2002). Developments in RNA chemistry, a personal view. Biochimie, 84:841–848.
11.
BennettCF and SwayzeEE. (2010). RNA Targeting Therapeutics: molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic Platform. Annu Rev Pharmacol Toxicol, 50:259–293.
HarborthJ, ElbashirSM, VandenburghK, ManningaH, ScaringeSA, WeberK 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.
14.
LiP, SergueevaZA, DobrikovM and ShawBR. (2007). Nucleoside and oligonucleoside boranophosphates: chemistry and properties. Chem Rev, 107:4746–4796.
15.
SheehanD. (2003). Biochemical properties of phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides. Nucleic Acids Res, 31:4109–4118.
16.
YamadaCM, DellingerDJ and CaruthersMH. (2007). Synthesis and biological activity of phosphonocarboxylate DNA. Nucleosides Nucleotides Nucleic Acids, 26:539–546.
17.
FroehlerBC. (1986). Deoxynucleoside H-phosphonate diester intermediates in the synthesis of internucleotide phosphate analogues. Tetrahedron Lett, 27:5575–5578.
18.
MichelT, DebartF, HeitzF and VasseurJ-J. (2005). Highly Stable DNA Triplexes Formed with Cationic Phosphoramidate Pyrimidine α-Oligonucleotides. ChemBioChem, 6:1254–1262.
19.
BaileyCP, DagleJM and WeeksDL. (1998). Cationic oligonucleotides can mediate specificinhibition of gene expression in Xenopus oocytes. Nucleic Acids Res, 26:4860–4867.
20.
ChaturvediS, HornT and LetsingerRL. (1996). Stabilization of triple-stranded oligonucleotide complexes: use of probes containing alternating phosphodiester and stereo-uniform cationic phosphoramidate linkages. Nucleic Acids Res, 24:2318–2323.
21.
MaierMA, GuzaevAP and ManoharanM. (2000). Synthesis of chimeric oligonucleotides containing phosphodiester, phosphorothioate, and phosphoramidate linkages. Org Lett, 2:1819–1822.
22.
MichelT, Martinand-MariC, DebartF, LebleuB, RobbinsI and VasseurJ-J. (2003). Cationic phosphoramidate α-oligonucleotides efficiently target single-stranded DNA and RNA and inhibit hepatitis C virus IRES-mediated translation. Nucleic Acids Res, 31:5282–5290.
23.
DagleJM, LittigJL, SutherlandLB and WeeksDL. (2000). Targeted elimination of zygotic messages in Xenopus laevis embryos by modified oligonucleotides possessing terminal cationic linkages. Nucleic Acids Res, 28:2153–2157.
24.
LennoxKA, SabelJL, JohnsonMJ, MoreiraBG, FletcherCA, RoseSD, BehlkeMA, LaikhterAL, WalderJA and DagleJM. (2006). Characterization of modified antisense oligonucleotides in Xenopus laevis embryos. Oligonucleotides, 16:26–42.
25.
JainML, BruicePY, SzabóIE and BruiceTC. (2012). Incorporation of Positively Charged Linkages into DNA and RNA Backbones: a Novel Strategy for Antigene and Antisense Agents. Chem Rev, 112:1284–1309.
26.
De MesmaekerA, WaldnerA, AltmannK-H and WendebornS. (1995). Backbone modifications in oligonucleotides and peptide nucleic acid systems. Curr Opin Struct Biol, 5:343–355.
27.
HornT, ChaturvediS, BalasubramaniamTN and LetsingerRL. (1996). Oligonucleotides with alternating anionic and cationic phosphoramidate linkages: synthesis and hybridization of stereo-uniform isomers. Tetrahedron Lett, 37:743–746.
28.
EndoM and KomiyamaM. (1996). Novel phosphoramidite monomer for the site-selective incorporation of a diastereochemically pure phosphoramidate to oligonucleotide. J Org Chem, 61:1994–2000.
29.
LetsingerRL, SingmanCN, HistandG and SalunkheM. (1988). Cationic oligonucleotides. J Am Chem Soc, 110:4470–4471.
30.
FroehlerBC, NgPG and MatteucciMD. (1988). Phosphoramidate analogues of DNA: synthesis and thermal stability of heteroduplexes. Nucleic Acids Res, 16:4831–4839.
31.
JungPM, HistandG and LetsingerRL. (1994). Hybridization of Alternating Cationic/Anionic Oligonucleotides to RNA Segments. Nucleosides Nucleotides, 13:1597–1605.
32.
ShabarovaZA. (1970). Synthetic Nucleotide-peptides. In: Progress in Nucleic Acid Research and Molecular Biology. DavidsonJN, CohnWE, eds. New York, NY: Academic Press, pp 145–182.
33.
JuodkaBA and SmrtJ. (1974). Synthesis of diribonucleoside phospho-(P → N)-amino acid derivatives. Collect Czech Chem Commun, 39:963–968.
34.
TomaszJ and SimoncsitsA. (1981). On the stability of phosphodiester-amide internucleotide bond. Tetrahedron Lett, 22:3905–3908.
35.
TomaszJ and LudwigJ. (1984). Instability of the phosphodiester-amide interribonucleotide bond in neutral aqueous solution. Nucleosides Nucleotides, 3:45–60.
36.
DeleaveyGF, WattsJK, AlainT, RobertF, KalotaA, AishwaryaV, PelletierJ, GewirtzAM, SonenbergN and DamhaMJ. (2010). Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing. Nucleic Acids Res, 38:4547–4557.
37.
DeglaneG, AbesS, MichelT, PrévotP, VivesE, DebartF, BarvikI, LebleuB and VasseurJ-J. (2006). Impact of the guanidinium group on hybridization and cellular uptake of cationic oligonucleotides. ChemBioChem, 7:684–692.
38.
DagleJM and WeeksDL. (1996). Positively charged oligonucleotides overcome potassium-mediated inhibition of triplex DNA formation. Nucleic Acids Res, 24:2143–2149.
39.
IkedaH, FernandezR, WilkA, BarchiJJ, HuangX and MarquezVE. (1998). The effect of two antipodal fluorine-induced sugar puckers on the conformation and stability of the Dickerson-Drew dodecamer duplex. Nucleic Acids Res, 26:2237–2244.
40.
ChiuYL and RanaTM. (2003). siRNA function in RNAi: a chemical modification analysis. RNA, 9:1034–1048.
41.
AthertonFR, OpenshawHT and ToddAR. (1945). 174. Studies on phosphorylation. Part II. The reaction of dialkyl phosphites with polyhalogen compounds in presence of bases. A new method for the phosphorylation of amines. J Chem Soc. 660–663.
42.
Le CorreSS, BerchelM, Couthon-GourvèsH, HaeltersJ-P and JaffrèsP-A. (2014). Atherton–Todd reaction: mechanism, scope and applications. Beilstein J Org Chem, 10:1166–1196.
43.
WestmanE and StrombergR. (1994). Removal of t-butyldimethylsilyl protection in RNA-synthesis. Triethylamine trihydrofluoride (TEA, 3HF) is a more reliable alternative to tetrabutylammonium fluoride (TBAF). Nucleic Acids Res, 22:2430–2431.
44.
MisiuraK, PietrasiakD and StecWJ. (1995). Dithymidylyl-3′,5′-phosphorofluoridates: new synthesis and stability under solvolytic conditions. J Chem Soc Chem Commun. 613–614.
45.
BollmarkM and StawinskiJ. (1996). A facile access to nucleoside phosphorofluoridate, nucleoside phosphorofluoridothioate, and nucleoside phosphorofluoridodithioate monoesters. Tetrahedron Lett, 37:5739–5742.
46.
LesnikEA and FreierSM. (1995). Relative Thermodynamic Stability of DNA, RNA, and DNA:RNA hybrid duplexes: relationship with base composition and structure. Biochemistry, 34:10807–10815.
47.
ConteMR, ConnGL, BrownT and LaneAN. (1997). Conformational properties and thermodynamics of the RNA duplex r(CGCAAAUUUGCG)2: comparison with the DNA analogue d(CGCAAATTTGCG)2. Nucleic Acids Res, 25:2627–2634.
48.
BramsenJB, LaursenMB, NielsenAF, HansenTB, BusC, LangkjaerN, BabuBR, HojlandT, AbramovM, et al. (2009). A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity. Nucleic Acids Res, 37:2867–2881.
49.
BramsenJB and KjemsJ. (2012). Engineering Small Interfering RNAs by Strategic Chemical Modification. In: siRNA Design. TaxmanDebra J., ed. Totowa, NJ: Humana Press, pp 87–109.
50.
AllersonCR, SioufiN, JarresR, PrakashTP, NaikN, BerdejaA, WandersL, GriffeyRH, SwayzeEE and BhatB. (2005). Fully 2‘-Modified Oligonucleotide Duplexes with Improved in Vitro Potency and Stability Compared to Unmodified Small Interfering RNA. J Med Chem, 48:901–904.
51.
OsbornMF, AltermanJF, NikanM, CaoH, DidiotMC, HasslerMR, ColesAH and KhvorovaA. (2015). Guanabenz (Wytensin™) selectively enhances uptake and efficacy of hydrophobically modified siRNAs. Nucleic Acids Res, 43:8664–8672.
52.
AltermanJF, HallLM, ColesAH, HasslerMR, DidiotM-C, ChaseK, AbrahamJ, SottosantiE, JohnsonE, et al. (2015). Hydrophobically modified siRNAs silence huntingtin mRNA in primary neurons and mouse brain. Mol Ther Nucleic Acids, 4:e266.
53.
GearyRS, YuR, SiwkowskiA and LevinAA (2007). Pharmacokinetic/Pharmacodynamic Properties of Phosphorothioate 2′-O-(2-Methoxyethyl)-Modified Antisense Oligonucleotides in Animals and Man. In: Antisense Drug Technology. CrookeStanley T., ed. Boca Raton, FL: CRC Press, pp 305–326.
54.
KhvorovaA, ReynoldsA and JayasenaSD. (2003). Functional siRNAs and miRNAs exhibit strand bias. Cell, 115:209–216.
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