Synthesis and Evaluation of Neutral Phosphate Triester Backbone-Modified siRNAs

Article abstract of DOI:10.1021/acsmedchemlett.0c00232

Two unsymmetrical dinucleotide phosphate triesters were synthesized via transesterification from tris(2,2,2-trifluoroethyl) phosphate. The protected triesters were phosphytilated to generate phosphoramidites for solid-phase oligonucleotide synthesis. Neutral phenylethyl phosphate-modified short-interfering RNAs (siRNAs) were synthesized and evaluated for their gene-silencing ability, siRNA strand selection, and resistance to nucleases. These backbone-modified phosphate triester siRNAs offer many improvements compared to natural unmodified siRNAs.

Full text of DOI:10.1021/acsmedchemlett.0c00232

pubs.acs.org/acsmedchemlett
Letter
Synthesis and Evaluation of Neutral Phosphate Triester Backbone-
Modified siRNAs
Kouta Tsubaki, Matthew L. Hammill, Andrew J. Varley, Mitsuru Kitamura, Tatsuo Okauchi,
and Jean-Paul Desaulniers*
Cite This: https://dx.doi.org/10.1021/acsmedchemlett.0c00232
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Two unsymmetrical dinucleotide phosphate triesters were synthesized via transesterification from tris(2,2,2-
trifluoroethyl) phosphate. The protected triesters were phosphytilated to generate phosphoramidites for solid-phase oligonucleotide
synthesis. Neutral phenylethyl phosphate-modified short-interfering RNAs (siRNAs) were synthesized and evaluated for their gene-
silencing ability, siRNA strand selection, and resistance to nucleases. These backbone-modified phosphate triester siRNAs offer many
improvements compared to natural unmodified siRNAs.
KEYWORDS: Short interfering RNA, backbone modification, phosphate triester, off-target effect
hort-interfering RNAs (siRNAs) are gaining traction as the
Snext generation therapeutics due to recent successes in the
field.1,2 siRNAs are gene-silencing molecules that target the
mRNA with high specificity.3,4 In order for this to occur, one
of the strands from the duplex is loaded into the RNA-
induced-silencing-complex (RISC) to form an active RISC-
guide-RNA complex, which directly targets the mRNA of
interest.5 With the recent US FDA approval of Patisiran and
Givosiran, which target rare diseases, and more RNA-based
therapeutics in clinical trials, confidence and optimism in
oligonucleotide-based therapeutics is strong.6 However, these
approved siRNAs contain several older generation modifica-
tions, namely the 2′-fluoro and 2′-O-methyl modifications.7,8
Despite this recent success, the future of siRNA development
toward more common diseases such as cancer depends on
expanding the chemical subset for modifications. This is
necessary in order to overcome some persistent challenges in
siRNA design.
backbone, which has seen utility with not only siRNAs but also
other therapeutic oligonucleotides such as antisense oligonu-
cleotides.11 This modification, along with other backbone
derivatives, such as boranophosphates, still retains the negative
charge.12 Neutral backbone alternatives such as amides,
triazoles, and other noncharged functional groups have been
synthesized and studied within siRNAs.13−15 However, one
group that has not been particularly well researched are neutral
triester phosphate backbones as a phosphodiester mimic. Some
previous groups have reported the synthesis and utility of alkyl
phosphate backbone derivatives of oligonucleotides.16−20
However, these types of studies are scarce, and these types
of triester modifications have not seen use within siRNA
applications.
In this study, we synthesized a small library of siRNAs
bearing a neutral modified phenylethyl phosphotriester back-
Received: May 1, 2020
Accepted: June 9, 2020
Published: June 9, 2020
The natural negatively charged phosphodiester backbone of
RNA is beset with poor cell-membrane permeability and is a
substrate for ubiquitous ribonucleases, which rapidly degrade
the RNA to monomers and/or short oligomers.9 Many
different types of backbone modifications have been reported
for siRNAs.10 The most common type is the phosphorothioate
https://dx.doi.org/10.1021/acsmedchemlett.0c00232
© XXXX American Chemical Society
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
A

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
bone on the passenger strand of siRNA duplexes (Figure 1).
These siRNAs exhibit potent gene silencing and some offer
been observed to be a useful modification within siRNA
design.22,23 Due to the acidic proton (NH) of the nitrogenous
bases interfering with the transesterification of the phosphate
trifluoroethyl ester, we protected the O-4 of the pyrimidine
heterocycles to solve this problem. First, allyl protected
deoxythymidine and deoxyuridine 1−4 were prepared
according to Noyori’s protocol.24 Next, both tert-butyldime-
thylsilyl (TBS)-protected thymidine derivative 1 and 2′-
deoxyuridine derivative 2 were treated with tris(2,2,2-
trifluoroethyl) phosphate in the presence of 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU), respectively, and the
monosubstituted phosphate triesters 5 and 6 were obtained via
selective transesterification in high yield. Then, the mono-
substituted phosphate triester 5 was treated with lithium
alkoxides generated in situ with the DMT-protected thymidine
Figure 1. Chemical structural difference between the natural
phosphate backbone of RNA (top) and the modified phenylethyl
phosphotriester backbone used in this study (bottom).
t
derivative 3 and BuOLi. The reaction proceeded selectively,
and the desired dinucleotide derivative 7 was obtained in high
yield. The dinucleotide derivative 8 was obtained selectively
from uridine derivatives 4 and 6 by a manner similar to that of
7. The transesterification between dinucleotide derivatives 7
and 8 and lithium phenylethoxide afforded allyl protected
phosphotriester dinucleotides 9 and 10 respectively in high
yield. Deprotections of the TBS group with 3HF·Et3N and the
allyl groups with a palladium catalyst (Pd(Ph3)4) gave the
expected phosphotriester dinucleotide monoalcohols 13 and
14.
Once the monoalcohols 13 and 14 were formed,
phosphoramidites from 13 and 14 were attempted by reaction
with 2-cyanoethyl diisopropylchlorophosphoramidite in the
presence of triethylamine in THF (Scheme 2).25 However,
enhanced target specificity compared to unmodified siRNAs.
Furthermore, these modifications offer resistance to nucleases
as measured by nuclease stability assays.
To synthesize siRNAs with a neutral phenylethyl phospho-
triester backbone, we utilized our recently published method-
ology (Scheme 1).21 Our efforts were focused on synthesizing
a 4,4′-dimethoxytrityl (DMT)-protected pyrimidine dinucleo-
tide, with a free alcohol. We chose to use a deoxynucleoside in
our design rather than a ribonucleoside in order to reduce the
number of synthetic steps and because deoxynucleotides have
Scheme 1. Synthesis of DMT-Protected Phenylethyl
Phosphotriester Dinucleotides
Scheme 2. Synthesis of DMT-Protected Phenylethyl
Phosphotriester Dinucleotide Phosphoramidites
both these phosphoramidites were unstable and could not be
purified using standard methods. As such, a short column was
used to separate unreacted phosphitylating reagent, and the
solutions containing the intermediate were immediately used
for solid-phase RNA synthesis.
During the synthesis, we used commercially available
phenoxyacetyl (PAC) and TBS-protected phosphoramidites
so we could use ultramild conditions to deprotect the RNA
from the resin (30% aqueous NH4OH for 2 h at room
temperature followed by HF for 2.5 h). This was important so
we would not get loss of the phenylethyl group via elimination
via standard conditions (for example, overnight deprotection
of benzoyl protecting groups with an EMAM solution [1:1
methylamine 33% wt in ethanol and methylamine 40 wt %]).
Table 1 highlights the sequences with the modifications that
we generated. We purified the sequences by HPLC and
confirmed their masses by qTOF-MS (quadrupole time-of-
flight mass spectroscopy). To verify that the sequences
adopted typical A-form helices, we performed circular
dichroism (CD) on the duplexes. CD spectrometry confirmed
the formation of the A-form helices (see Supporting
B
https://dx.doi.org/10.1021/acsmedchemlett.0c00232
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
a
SiRNAs 2−5 all exhibit excellent dose-dependent gene-
silencing within the wild-type siRNA (wt) range (IC50s
ranging from 3.5−22.9 pM, see Figure S2 in the Supporting
Information). Therefore, these phenylethyl phosphotriester
backbone modifications are suitable substrates for the RNA
interference pathway.
Table 1. Sequence of siRNAs and Melting Temperatures
(Tm)
siRNA
siRNA sequence
Tm (° C)
wt
5′-CUUACGCUGAGUACUUCGAdTdT-3′
3′-dTdTGAAUGCGACUCAUGAAGCU-5
5′-CdUxdUACGCUGAGUACdUxdUCGAdTdT-3′
3′-dTdTGAAUGCGACUCAUGAAGCU-5
5′ - CUUACGCUGAGUACUUCGAdUxdU - 3′
3′-dTdTGAAUGCGACUCAUGAAGCU-5
5′-CUUACGCUGAGUACUUCGAdUxdUdT-3′
3′-dTdTGAAUGCGACUCAUGAAGCU-5
5′-CUUACGCUGAGUACUUCGAdTxdT-3′
3′-dTdTGAAUGCGACUCAUGAAGCU-5
5′-CUUACGCUGAGUACUUCGAdTxdTdT-3′
3′-dTdTGAAUGCGACUCAUGAAGCU-5
74.1
1
2
3
4
5
71.8
74.4
77.1
73.4
75.1
We next examined the impact of the phenylethyl
phosphotriester modification on RISC strand selection through
the characterization of select siRNAs. We recently published a
paper outlining a quantitative reverse transcriptase polymerase
chain reaction (qRT-PCR) method to quantify the relative
amounts of guide- and passenger-mediated gene-silencing of
our siRNA.30 In unmodified siRNA, the guide strand is
preferentially incorporated into the RISC (by siRNA design)
due to the preference for (1) nucleotide preference for A or U
at the 5′ end and (2) the strand with lower 5′ thermodynamic
stability.5,28,29 While these considerations lower passenger
strand uptake, the risk of passenger strand associated off-target
effects encourages the development of siRNA with further
improved selectivity. Therefore, this assay was used to compare
the strand selection of wt siRNA and modified siRNAs 1−3
developed in this study. As expected, the guide strand was
significantly more active in wt siRNA than the passenger strand
(Figure 3). At 8 pM, the guide strand reached a maximum
silencing plateau of ∼75%, whereas the passenger strand
required up to 800 pM to exert the same effect. The addition
of the phenylethyl triphosphate modifications to wt siRNA, as
seen in siRNAs 1−3, significantly altered the activity of the two
strands. In agreement with the dual-luciferase reporter assays,
the guide strand of siRNAs 1−3 showed reduced activity with
1 being the least active. Remarkably, when compared to wt
siRNA, guide strand selection was enhanced for siRNA 1 but
diminished for siRNAs 2 and 3. We predict that the improved
selection of siRNA 1 is due to the inability of the nonionic
phenylethyl triphosphate to mimic the natural charged
backbone interactions that occur between the phosphate and
amino acid residues of the RISC.31 In agreement with previous
research using this assay, heightened selection of the passenger
strand is associated with chemical modifications at the 3′
terminal of the passenger strand.30 The ability for 3′
a
dTxdT corresponds to the deoxythymidine-based modification;
dUxdU corresponds to the deoxyuridine-based modification. The top
strand is the passenger strand; the bottom strand is the guide strand.
The guide strand is 5′-phosphorylated.
Information Figure S-1). In addition, we examined their
thermal stability by measuring their melting temperature (Tm).
In general, no large changes in Tm were observed compared to
wt siRNA. This suggests that the impact of the phosphotriester
backbone modification on the duplex’s thermal stability is
minimal.
We designed sequences that target firefly luciferase. Briefly,
expression plasmids individually coding for firefly and Renilla
luciferase (internal control), and the desired siRNA was
incubated with lipofectamine, and the mixture was transfected
to mammalian cells. Cells were then lysed 24 h post
transfection and the efficacy of the siRNAs was evaluated
using the Dual-Luciferase Reporter Assay (Promega) system.
siRNA 1 contains the modifications at two internal locations
within the passenger strand. As shown in Figure 2, excellent
dose-dependent gene silencing is observed with siRNA 1
exhibiting an IC50 of 66.9 pM. Compared to wild-type siRNA
(wt), this exhibits a small loss of potency (IC50 = 5 pM).
However, this dose-dependent knockdown is comparable to
other reports with internal neutral backbone modifications.26,27
Figure 2. Reduction in firefly luciferase protein expression related to the potency of phenylethyl phosphate-modified siRNAs using the dual-
luciferase reporter assay. The siRNAs were tested between 10 and 800 pM, with firefly luciferase expression normalized to Renilla luciferase. *
indicates statistical significance by t test analysis (p < 0.05, n = 3). Inset: Sequence and location of modifications used in chemically modified
siRNA.
C
https://dx.doi.org/10.1021/acsmedchemlett.0c00232
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
selection.33 siRNA 1 also carries a neutral linkage at the 5′
terminal of the passenger strand and may be selected in a
similar manner. Therefore, electrostatic interactions between
the first 5′ linkage and the RISC likely play a significant role in
strand selection, which is not surprising given the phosphate
dependent RNA−protein interactions identified at this
location within the RISC.34
Finally, we examined the effect of nuclease stability on these
siRNAs. It is well-known that RNAs exhibit high degradation
rates in the presence of nuclease-rich environments.35
Unmodified (wt siRNA) and modified siRNAs 1−5 were
incubated in fetal bovine serum (FBS) for up to 4 h to analyze
the degraded products in a time-dependent manner using
polyacrylamide gel electrophoresis (Figure 4).
Unmodified wt siRNA degraded almost immediately, with
almost no full-length siRNA remaining after 0.5 h. siRNA 1,
which contains the internal backbone modifications, also
degraded almost immediately, with little remaining after 1 h. In
contrast, siRNA 2, which contains the phenylethyl backbone
modification dUxdU (the deoxyuridine-based dinucleotide),
directly at the 3′-end of the passenger strand, exhibited
excellent nuclease stability up to 4 h. siRNA 3, which contains
the backbone modification dUxdU directly adjacent to a single
deoxythymidine nucleoside residue on the 3′-end overhang,
also exhibits enhanced nuclease stability compared to wt
siRNA (intact siRNA viewed as long as 1 h). However,
compared to siRNA 2, this one is degraded more rapidly.
Finally, siRNAs 3 and 4 contain the phenylethyl backbone
modification dTxdT (the deoxythymidine-based dinucleotide)
at the 3′-end and exhibit good nuclease stability up to 2 and 3
h, respectively. Thus, 3′-end phenylethyl phosphate backbone
modifications offer enhanced nuclease resistance and thus may
be used as a viable option for increasing the lifetime of siRNAs
Figure 3. Reduction in the appropriate firefly luciferase variant mRNA
due to guide and passenger strand activity. Guide and passenger
strand target knockdown by wildtype and phenylethyl phosphate-
modified siRNAs was measured by qPCR analysis. Target expression
was normalized against the Renilla luciferase expression control.
*indicates statistical significance by t test analysis (p < 0.05, n = 3).
modifications to encourage passenger strand uptake suggests
that similar modifications may be well tolerated in the guide
strand and warrants further investigation.
Many other phosphate modifications have been developed,
yet few have been investigated in siRNA. Negatively charged
backbone modifications such as the phosphorodithioate,32 and
neutral ones, such as triazole,14 and amide linkages13,26 have
been used to improve serum stability and/or cell permeability.
The location of these modifications also plays a major role in
whether it is RNAi-activating or deactivating. For example, the
placement of a single amide linkage at the 5′ terminal of the
passenger strand was dramatically effective at improving strand
Figure 4. Nuclease stability assay. Samples were run on 20% nondenaturing polyacrylamide gel degradation products of siRNAs wt and 1−5 after
incubation with 13.5% fetal bovine serum at 37 °C from 0 to 4 h.
D
https://dx.doi.org/10.1021/acsmedchemlett.0c00232
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
in the blood serum. Internal modifications (siRNA 1) do not
appear to offer a significant advantage in stability.
AUTHOR INFORMATION
■
Corresponding Author
In conclusion, we are reporting the synthesis of a neutral
phenylethyl phosphosphate triester DMT-phosphoramidite
using recently published methodology published from our
lab that involves transesterification from tris (2,2,2-trifluor-
oethyl phosphate).21 From this phosphoramidite, a small
library of siRNAs bearing this modification at different
positions of the passenger strand was generated. Excellent
gene-silencing was observed as monitored using the Dual-
Luciferase Reporter Assay. Furthermore, we examined the
guide- and passenger-strand mediated gene silencing and
observed that siRNA 1, which contains two internal phenyl-
ethyl phosphate modifications improved the desired guide
strand-mediated gene silencing, whereas the 3′-end modifica-
tions appeared to not offer any significant advantage in
comparison to wt siRNA. In contrast, siRNAs 2−5, which
contained the 3′-end modifications, offered enhanced nuclease
stability compared to internal backbone modifications (siRNA
1). Phosphate triesters have been studied for many years, but
to our knowledge these specific neutral phosphate triester
compounds have not been tested for gene silencing or nuclease
stability, and thus, we believe that this is the first type of
investigation. The passenger strand was selected for
modification because we wanted to separate the gene silencing
effects from the nuclease stability effects. Our choice in using
the O-phenylethyl phosphate was based on our desire to
incorporate an aromatic hydrophobic functionality, while
maintaining a relatively short aliphatic carbon chain to the
phosphate backbone. Future work involves expanding and
examining the synthesis of several different kinds of derivatives
with variable length alkyl chains, and bulkier groups to further
examine new structure−activity relationships.
Jean-Paul Desaulniers − Faculty of Science, University of
Ontario Institute of Technology, Oshawa, Ontario L1G 0C5,
Canada; orcid.org/0000-0002-9596-4552; Email: Jean-
Paul.Desaulniers@ontariotechu.ca
Authors
Kouta Tsubaki − Department of Applied Chemistry, Graduate
School of Engineering, Kyushu Institute of Technology,
Kitakyushu 804-8550, Japan; Faculty of Science, University of
Ontario Institute of Technology, Oshawa, Ontario L1G 0C5,
Canada
Matthew L. Hammill − Faculty of Science, University of Ontario
Institute of Technology, Oshawa, Ontario L1G 0C5, Canada
Andrew J. Varley − Faculty of Science, University of Ontario
Institute of Technology, Oshawa, Ontario L1G 0C5, Canada
Mitsuru Kitamura − Department of Applied Chemistry,
Graduate School of Engineering, Kyushu Institute of Technology,
Kitakyushu 804-8550, Japan
Tatsuo Okauchi − Department of Applied Chemistry, Graduate
School of Engineering, Kyushu Institute of Technology,
Kitakyushu 804-8550, Japan; orcid.org/0000-0002-0143-
149X
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00232
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge Natural Sciences and Engineering Research
Council (NSERC) and the Institute of Rheological Functions
of Food in Japan for funding.
Modification of the guide strand could have made silencing
less effective, and therefore the siRNA overall would have had
less utility than it currently does. This way, the silencing is
effective, but the phosphate triester provides high nuclease
resistance as well. This provides the benefit of selecting a
phosphate triester for nuclease stability but then leaves the
guide strand open to further potential modification if desired.
Thus, it appears that the activity and stability of these novel
backbone-modified siRNAs can be fine-tuned depending on its
location within the passenger strand. Future directions include
developing oligonucleotides with other triphosphate modifica-
tions such as small molecules that can further explore the
structure−activity relationship of oligonucleotides.
REFERENCES
■
(1) de Paula Brandao
Almeida, R. 2019 FDA TIDES (Peptides and Oligonucleotides)
Harvest. Mol. Diagn. Ther. 2020, 24, 61.
̃
, P. R.; Titze-De-Almeida, S. S.; Titze-De-
(2) Ruger, J.; Ioannou, S.; Castanotto, D.; Stein, C. A.
̈
Oligonucleotides to the (Gene) Rescue: FDA Approvals 2017−
2019. Trends Pharmacol. Sci. 2020, 41, 27.
(3) Fire, A.; Xu, S. Q.; Montgomery, M. K.; Kostas, S. A.; Driver, S.
E.; Mello, C. C. Potent and specific genetic interference by double-
stranded RNA in. Nature 1998, 391, 806.
(4) Matranga, C.; Tomari, Y.; Shin, C.; Bartel, D. P.; Zamore, P. D.
Passenger-strand cleavage facilitates assembly of siRNA into Ago2-
containing RNAi enzyme complexes. Cell 2005, 123, 607.
(5) Khvorova, A.; Reynolds, A.; Jayasena, S. D. Functional siRNAs
and miRNAs exhibit strand bias. Cell 2003, 115, 209.
(6) Setten, R. L.; Rossi, J. J.; Han, S.-P. The current state and future
directions of RNAi-based therapeutics. Nat. Rev. Drug Discovery 2019,
18, 421.
(7) Manoharan, M. 2′-Carbohydrate modifications in guide
oligonucleotide therapy: importance of conformation, configuration
and conjugation. Biochim. Biophys. Acta, Gene Struct. Expression 1999,
1489, 117.
(8) Pallan, P. S.; Greene, E. M.; Jicman, P. A.; Pandey, R. K.;
Manoharan, M.; Rozners, E.; Egli, M. Unexpected origins of the
enhanced pairing affinity of 2′-fluoro-modified RNA. Nucleic Acids
Res. 2011, 39, 3482.
(9) Wang, J.; Lu, Z.; Wientjes, M. G.; Au, J. L. S. Delivery of siRNA
therapeutics: barriers and carriers. AAPS J. 2010, 12, 492.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00232.
Synthetic procedures for compounds 5−14 (pages S3−
S10), all procedures for oligonucleotide synthesis and
characterization (pages S11−S12), procedures for
nuclease stability testing (page S13), and procedures
for testing gene silencing ability in vitro (pages S13−
1
S14). In addition, H/13C/19F/31P NMR spectral data
for compounds 5−14 (pages S18−S33) (PDF)
E
https://dx.doi.org/10.1021/acsmedchemlett.0c00232
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
(10) Shen, X.; Corey, D. R. Chemistry, mechanism and clinical
status of guide oligonucleotides and duplex RNAs. Nucleic Acids Res.
2018, 46, 1584.
(28) Noland, C. L.; Doudna, J. A. Multiple sensors ensure guide
strand selection in human RNAi pathways. RNA 2013, 19, 639−648.
́
(29) Schwarz, D. S.; Hutvagner, G.; Du, T.; Xu, Z.; Aronin, N.;
Zamore, P. D. Asymmetry in the assembly of the RNAi enzyme
(11) Eckstein, F. Phosphorothioates, essential components of
therapeutic oligonucleotides. Nucleic Acid Ther. 2014, 24, 374.
(12) Hall, A. H. S.; Wan, J.; Shaughnessy, E. E.; Ramsay Shaw, B.;
Alexander, K. A. RNA inferference using boranophosphate siRNAs:
structure-activity relationships. Nucleic Acids Res. 2004, 32, 5991.
(13) Mutisya, D.; Selvam, C.; Lunstad, B. D.; Pallan, P. S.; Haas, A.;
Leake, D.; Egli, M.; Rozners, E. Amides are excellent mimics of
phosphate internucleoside linkages and are well tolerated in short
interfering RNAs. Nucleic Acids Res. 2014, 42, 6542.
(14) Efthymiou, T. C.; Huynh, V.; Oentoro, J.; Peel, B.; Desaulniers,
J.-P. Efficient synthesis and cell-based silencing activity of siRNAs that
contain triazole backbone linkages. Bioorg. Med. Chem. Lett. 2012, 22,
1722.
complex. Cell 2003, 115, 199−208.
(30) Varley, A. J.; Hammill, M. H.; Salim, L.; Desaulniers, J.-P.
Effects of Chemical Modifications on siRNA Strand Selection in
Mammalian Cells. Nucleic Acid Ther. 2020, DOI: 10.1089/
nat.2020.0848.
(31) Schirle, N. T.; MacRae, I. J. The crystal structure of human
Argonaute2. Science 2012, 336, 1037.
(32) Yang, X.; Sierant, M.; Janicka, M.; Peczek, L.; Martinez, C.;
Hassell, T.; Li, N.; Li, X.; Wang, T.; Nawrot, B. Gene silencing activity
of siRNA molecules containing phosphorodithioate substitutions.
ACS Chem. Biol. 2012, 7, 1214−1220.
(33) Hardcastle, T.; Novosjolova, I.; Kotikam, V.; Cheruiyot, S. K.;
Mutisya, D.; Kennedy, S. D.; Egli, M.; Kelley, M. L.; van Brabant
Smith, A.; Rozners, E. A Single Amide Linkage in the Passenger
Strand Suppresses Its Activity and Enhances Guide Strand Targeting
of siRNAs. ACS Chem. Biol. 2018, 13, 533−536.
(34) Elkayam, E.; Kuhn, C. D.; Tocilj, A.; Haase, A. D.; Greene, E.
M.; Hannon, G. J.; Joshua-Tor, L. The structure of human argonaute-
2 in complex with miR-20a. Cell 2012, 150, 100−110.
(35) Bertrand, J.-R.; Pottier, M.; Vekris, A.; Opolon, P.;
Maksimenko, A.; Malvy, C. Comparison of guide oligonucleotides
and siRNA in cell culture and in vivo. Biochem. Biophys. Res. Commun.
2002, 296, 1000.
(15) Efthymiou, T. C.; Peel, B.; Huynh, V.; Desaulniers, J. P.
Evaluation of siRNAs that contain internal variable-length spacer
linkages. Bioorg. Med. Chem. Lett. 2012, 22, 5590.
́
(16) Gutierrez-Puente, Y.; Tari, A. M.; Ford, R. J.; Tamez-Guerra,
R.; Mercado-Hernandez, R.; Santoyo-Stephano, M.; Lopez-Berestein,
G. Cellular pharmacology of P-ethoxy guide oligonucleotides targeted
to Bcl-2 in a follicular lymphoma cell line. Leuk. Lymphoma 2003, 44,
1979.
́
(17) Gutierrez-Puente, Y.; Tari, A. M.; Stephens, C.; Rosenblum,
M.; Guerra, R. T.; Lopez-Berestein, G. Safety, pharmacokinetics and
tissue distribution of liposomal P-ethoxy guide oligonucleotides
targeted to Bcl-2. J. Pharmacol. Exp. Ther. 1999, 291, 865.
(18) Miller, P. S.; Fang, K. N.; Kondo, N. S.; Paul, O. P.; Ts’o, P. O.
P. Conformation and interaction of dinucleoside mono- and
diphosphates. V. Syntheses and properties of adenine and thymine
nucleoside alkyl phosphotriesters, the neutral analogs of dinucleoside
monophosphates. J. Am. Chem. Soc. 1971, 93, 6657−6665.
(19) Koole, L. H.; Moody, H. M.; Broeders, N. L. H. L.; Quaedflieg,
P. J. L. M.; Kuijpers, W. H. A.; van Genderen, M. H. P.; Coenen, A. J.
J. M.; Sjoerd van der Wal, S.; Buck, H. M. Synthesis of Phosphate-
Methylated DNA Fragments Using 9-Fluorenylmethoxycarbonyl as
Transient Base Protecting Group. J. Org. Chem. 1989, 54, 1657.
(20) Dohno, C.; Shibata, T.; Okazaki, M.; Makishi, S.; Nakatani, K.
Amphiphilic DNA Duplex Stabilized by a Hydrophobic Zipper. Eur. J.
Org. Chem. 2012, 2012, 5317−5323.
(21) Tsubaki, K.; Shimooka, H.; Kitamura, M.; Okauchi, T. Selective
transesterification of 2,2,2-trifluoroethyl phosphates: synthesis of
mixed unsymmetrical phosphates. Org. Lett. 2019, 21, 9779.
(22) Hogrefe, R. I.; Lebedev, A. V.; Zon, G.; Pirollo, K. F.; Rait, A.;
Zhou, Q.; Yu, W.; Chang, E. H. Chemically modified short interfering
hybrids (siHYBRIDS): nanoimmunoliposome delivery in vitro and in
vivo for RNAi of HER-2. Nucleosides, Nucleotides Nucleic Acids 2006,
25, 889.
(23) Ui-Tei, K.; Naito, Y.; Zenno, S.; Nishi, K.; Yamato, K.;
Takahashi, F.; Juni, A.; Saigo, K. Functional dissection of siRNA
sequence by systematic DNA substitution: modified siRNA with a
DNA seed arm is a powerful tool for mammalian gene silencing with
significantly reduced off-target effect. Nucleic Acids Res. 2008, 36,
2136.
(24) Hayakawa, Y.; Hirose, M.; Noyori, R. O-Allyl protection of
guanine and thymine residues in oligodeoxyribonucleotides. J. Org.
Chem. 1993, 58, 5551.
(25) Caruthers, M. H. Gene synthesis machines: DNA chemistry
and its uses. Science 1985, 230, 281.
(26) Gong, W.; Desaulniers, J. P. Gene-silencing properties of
siRNAs that contain internal amide-bond linkages. Bioorg. Med. Chem.
Lett. 2012, 22, 6934.
(27) Mutisya, D.; Hardcastle, T.; Cheruiyot, S. K.; Pallan, P. S.;
Kennedy, S. D.; Egli, M.; Kelley, M. L.; Smith, A. van B.; Rozners, E.
Amide linkages mimic phosphates in RNA interactions with proteins
and are well tolerated in the guide strand of short interfering RNAs.
Nucleic Acids Res. 2017, 45, 8142.
F
https://dx.doi.org/10.1021/acsmedchemlett.0c00232
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX