Building siRNAs with Cubes: Synthesis and Evaluation of Cubane-Modified siRNAs

Article abstract of DOI:10.1002/cbic.202100334

Cubane molecules hold great potential for medicinal chemistry applications due to their inherent stability and low toxicity. In this study, we report the synthesis of a cubane derivative phosphoramidite for the incorporation of cubane into small interfering RNAs (siRNAs). Synthetic siRNAs rely on chemical modifications to improve their pharmacokinetic profiles. However, they are still able to mediate sequence-specific gene silencing via the endogenous RNA interference pathway. We designed a library of siRNAs bearing cubane at different positions within the sense and antisense strands. All siRNAs showed excellent gene-silencing activity, with IC₅₀ values ranging from 45.4 to 305 pM. Incorporating the cubane modification in both the sense and antisense strand led to viable duplexes with good biological activity. To the best of our knowledge, this is the first report of siRNAs bearing a cubane derivative within the backbone.

Full text of DOI:10.1002/cbic.202100334

Combining Chemistry and Biology
Accepted Article
Title: Building siRNAs with cubes: synthesis and evaluation of cubane-
modified siRNAs
Authors: Matthew L. Hammill, Lidya Salim, Kouta Tsubaki, Andrew J.
Varley, Mitsuru Kitamura, Tatsuo Okauchi, and Jean-Paul
Desaulniers
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.202100334
Link to VoR: https://doi.org/10.1002/cbic.202100334
01/2020

10.1002/cbic.202100334
ChemBioChem
RESEARCH ARTICLE
Building siRNAs with cubes: synthesis and evaluation of cubane-
modified siRNAs
Matthew L. Hammill,[a] Lidya Salim,[a] Kouta Tsubaki,[a,b] Andrew J. Varley,[a] Mitsuru Kitamura,[b] Tatsuo
Okauchi,[b] and Jean-Paul Desaulniers[a]*
[a]
M. L. Hammill, L. Salim, K. Tsubaki, A. Varley, J.-P. Desaulniers*
Faculty of Science
University of Ontario Institute of Technology
2000 Simcoe Street North, Oshawa, Ontario L1G 0C5 Canada
E-mail: Jean-Paul.Desaulniers@ontariotechu.ca
K. Tsubaki, M. Kitamura, T. Okauchi
[b]
Department of Applied Chemistry, Graduate School of Engineering
Kyushu Institute of Technology
1-1 Sensui-cho, Tobata, Kitakyushu 804-8550, Japan
Supporting information for this article is given via a link at the end of the document.
1
2
3
4
5
6
7
8
9
Abstract: Cubane molecules hold great potential for medicin4al0 anionic charge of the RNA molecule. This has led to the
chemistry applications due to its inherent stability and low toxicity. 4In1 development of neutral backbone replacements such as
this manuscript, we report the synthesis of a cubane derivativ4e2 amides,[8] triazoles,[9] neutral phosphate triesters,[10] and aromatic
phosphoramidite for the incorporation of cubane into small interferin4g3 scaffolds.[11] While this is an important limitation to overcome, it
RNAs (siRNAs). Synthetic siRNAs rely on chemical modifications 4to4 bears mentioning that this work is focused on the compatibility of
improve their pharmacokinetic profiles. However, they are still able 4to5 the cubane moiety in the RISC complex, not necessarily
mediate sequence-specific gene silencing via the endogenous RN4A6 improving the pharmacokinetic properties of the siRNA at this
interference pathway. We designed a library of siRNAs bearing th4e7 stage. With this in mind, we present the following results on the
cubane at different positions within the sense and antisense strand4s.8 compatibility of cubane modifications within the RNAi pathway.
10 All siRNAs showed excellent gene-silencing activity, with IC50 values
11 ranging from 45.4 to 305 pM. Incorporating the cubane modification
12 in both the sense and antisense strand led to viable duplexes with
13 good biological activity. To the best of our knowledge, this is the first
14 report of siRNAs bearing a cubane derivative within the backbone.
49 Cubane is a saturated hydrocarbon system, with a three-
This synthetically
50 dimensional cube-like arrangement.
51 challenging molecule has been of great interest in theoretical
52 organic chemistry and, more recently, has become a promising
53 molecule for medicinal chemistry applications.[12] Cubane can act
54 as a bioisostere for benzene, but it has the added benefit of being
55 less toxic and more biologically stable.[13] As a result, there is
15 Introduction
56 increasing interest in the synthesis of novel, cubane-bearing small
57 molecules that could maintain the same functionality as the
16 Short-interfering RNAs (siRNAs) belong to a class of double-
17 stranded RNA molecules that can induce gene silencing via the
58 previous generation of benzene-based therapies. One such case
59 is the use of a cubane derivative in a nucleic acid aptamer for
18 RNA interference (RNAi) pathway.[1] The siRNA is
19
fir6st0 selective recognition of malaria biomarkers.[14] Cubane molecules
incorporated into an RNA-induced silencing complex (RISC) as6a1 also share a similar reactivity profile to benzene, in that they
20 duplex, but one of the strands, called the passenger strand must
62 undergo substitution reactions despite them not being aromatic
be removed for proper RNAi activity. The guide strand, which 6is3 themselves.[15] Therefore, they can be further modified with any
21
22 complementary to the target mRNA, remains bound to the active
23 complex and directs mRNA cleavage to achieve sequence-
24 specific gene silencing.[2] Recently, these biomolecules have
25 gained widespread interest due to the US FDA-approval of three
64 number of substituents depending on the desired application.
65 In this study, we employed a reported synthetic approach to
66 prepare a cubane ethoxy derivative which was then used as the
siRNA-based formulations for the treatment of rare genet6ic7 starting material to synthesize a novel cubane derivative
26
27 conditions.[3] Despite this progress, there are many limitations
28 associated with the inherent structure of siRNAs, so they must be
29 chemically modified to improve their pharmacokinetic profiles.
68 phosphoramidite for oligonucleotide synthesis. We designed a
69 small library of siRNAs bearing a single cubane derivative at
70 different positions within the sense or antisense strand (Figure 1).
Several chemical modifications, such as 2’-fluoro and 2’-O-meth7yl1, We then investigated the gene-silencing activity of duplexes
30
31 have been reported and extensively used for both in vitro and
32 clinical applications.[4-6] While these modifications have been
33 instrumental in opening the field up to new investments, the future
34 and pace of siRNA development will depend on how quickly novel
35 chemical modifications can be synthesized, tested, and applied
36 within biological systems.
72 modified at either the sense strand, the antisense strand, or both.
73 All duplexes adopted the appropriate A-form helical conformation
74 required for incorporation into the RISC. Most siRNAs induced
75 potent gene silencing activity and offered some enhanced target
76 specificity compared to unmodified wildtype siRNA.
77
37 A common strategy involves replacing the phosphate backbone
38 with phosphorothioate or boranophosphate to increase nuclease
39 resistance.[7] However, these modifications do not reduce the
1
This article is protected by copyright. All rights reserved.

10.1002/cbic.202100334
ChemBioChem
RESEARCH ARTICLE
105 liquid chromatography (HPLC) (Supplementary Figure S-1) and
106 characterized by quadrupole time-of-flight mass spectroscopy
107 (qTOF-MS). siRNA synthetic yields were >99% based on the
108 colour of the DMT cation that was removed at each step, using
109 standard synthetic procedures (See supplement S1 general
110 section for synthetic parameters). The cubane modification was
111 placed at several locations of interest. Modifications at the 3’ end
112 are generally well tolerated and were included to investigate
113 whether the cubane modification dramatically impacts siRNA
114 activity. Modification of the seed region (nucleotides 2 to 8 from
78
79
Figure 1. Structure of RNA with native phosphate backbone (top) and1th1e5 the 5’ end) can dramatically reduce activity of the associated
80 cubane modification (bottom).
116 strand as it interrupts primary target recognition. Thus, the cubane
117 was only placed within the seed region of the sense strand.
118 Central modifications may prevent maturation of the RISC and/or
119 reduce the ability for the mature RISC to cleave target mRNA
120 strands. The impact of the cubane within the central region was
121 therefore explored on both the antisense and sense strand.The
81 Results and Discussion
82 To synthesize siRNAs incorporating the 1,4-ethoxy cubane
derivative, we followed a standard cubane synthesis proced1ur2e2 resulting sequences are summarized in Table 1 and mass
83
84 developed by the Tsanaktsidis lab.[15,16]
This was followed by 1th2e3 spectroscopy data can be found in the Supplementary Table S-1.
85 reduction of the acetylated diol, monoprotection using
124 Sense and antisense strands were annealed prior to biophysical
86 dimethoxytrityl chloride (DMT-Cl), and phosphitylation of the
125 and biological assays. We prepared twelve siRNA duplexes, with
87 DMT-protected monoalcohol for incorporation into siRNAs
(Scheme 1). We focused on developing a symmetrical 1,4 etho1xy2-6 siRNAs 1-4 containing the cubane modification within the sense
88
127 strand, siRNAs 5 and 6 containing the cubane modification within
89 cubane derivative that could be used as a backbone replacem1en2t8 the antisense strand and siRNAs 7-12 being modified on both
for benzene. Starting with the acetylated cubane, reduction of 1th2e9 strands. To confirm that the resulting duplexes adopted the active
90
91 esters with lithium aluminum hydride in tetrahydrofuran (THF)
130 A-form helical conformation recognized by the RISC, circular
92 yielded the 1,4-ethoxy cubane derivative 1 (62%). Next, diol 1 was
131 dichroism (CD) spectroscopy was performed (See Figure S2 in
93 treated with DMT-Cl in dichloromethane (DCM) to give the singly
132 supplement S1). All siRNAs, including those that were modified
94 protected monoalcohol 2 (47%), which was then reacted with 2-
133 on both the sense and antisense strand (siRNAs 7-12) formed
95 cyanoethyl diisopropylchlorophosphoramidite in the presence of
134 viable duplexes and adopted the desired conformation
triethylamine in DCM. The resulting phosphoramidite 3 represe1nt3s5 (Supplementary Figure S-2). Since the potency of siRNAs can be
96
97 our solid-phase synthesis building block, which was employed to
136 modulated by their thermodynamic properties, we also evaluated
98 prepare a library of siRNAs. To the best of our knowledge, this is
137 the thermal stability of each duplex. The melting temperature (Tm)
99 the first report of an siRNA bearing a cubane replacement within
138 data is summarized in Table 1.
100 its backbone.
139 Table 1. Sequences, melting temperatures and IC50 values of anti-luciferase
140 cubane siRNAs
siRNA
Sequence
Tm
(°C)
Δ Tm
(°C)
IC50 ± SE
(pM)
5’ CUU ACG CUG AGU ACU UCG ATT 3’
3’ TTG AAU GCG ACU CAU GAA GCU 5’
wt
1
76
72
72
71
63
65
72
69
63
65
61
-
5.9 ± 2.3
5’ CUU ACG CUG AGU ACU UCG ATX 3’
3’ TTG AAU GCG ACU CAU GAA GCU 5’
-4
83.9 ± 46.7
45.4 ± 11.7
108.8 ± 11.7
152.5 ± 46.3
305.5 ± 6.5
101.9 ± 97.9
754.3 ± 10.2
816.0 ± 20.5
1259 ± 410.8
557.7 ± 68.8
5’ CUU ACG XUG AGU ACU UCG ATT 3’
3’ TTG AAU GCG ACU CAU GAA GCU 5’
2
-4
5’CUU ACG CUG AXU ACU UCG ATT -3’
3’ TTG AAU GCG ACU CAU GAA GCU 5’
3
-5
5’ CUU ACG CUG AGU ACU UCG AX 3’
3’ TTG AAU GCG ACU CAU GAA GCU 5’
4
-13
-11
-4
5’ CUU ACG CUG AGU ACU UCG ATT 3’
3’ TTG AAU GCG XCU CAU GAA GCU 5’
5
5’ CUU ACG CUG AGU ACU UCG ATT 3’
3’ XG AAU GCG ACU CAU GAA GCU 5’
6
5’ CUU ACG CUG AGU ACU UCG ATX 3’
3’ TTG AAU GCG XCU CAU GAA GCU 5’
7
-7
5’ CUU ACG CUG AGU ACU UCG ATX 3’
3’ XG AAU GCG ACU CAU GAA GCU 5’
101
8
-13
-11
-15
102 Scheme 1. Synthesis of cubane DMT-protected phosphoramidite.
5’ CUU ACG XUG AGU ACU UCG ATT 3’
3’ TTG AAU GCG XCU CAU GAA GCU 5’
9
103 After standard oligonucleotide synthesis, cleavage and
104 deprotection, RNA strands were purified by high-performance
5’ CUU ACG XUG AGU ACU UCG ATT 3’
3’ XG AAU GCG ACU CAU GAA GCU 5’
10
2
This article is protected by copyright. All rights reserved.

10.1002/cbic.202100334
ChemBioChem
RESEARCH ARTICLE
145 luciferase (internal control). HeLa cells were transfected with both
573.11 ±41610.6plasmids as well as the appropriate siRNA using the transfection
4361.74±722.2 reagent Lipofectamine 2000TM. Cells were lysed 24 hours post-
148 transfection after which the Dual-Luciferase Reporter Gene Assay
5’ CUU ACG CUG AXU ACU UCG ATT 3’
3’ TTG AAU GCG XCU CAU GAA GCU 5’
11
52
61
-24
-15
5’ CUU ACG CUG AXU ACU UCG ATT 3’
3’ XG AAU GCG ACU CAU GAA GCU 5’
12
149
The top strand corresponds to the sense strand. The bottom strand corresponds to the antisense strandw. Xas performed following the manufacturer’s procedure. We first
corresponds to the cubane moiety. The antisense strand is phosphorylated at the 5’ end.
150 investigated the biological activity of the siRNAs modified on
151 either the sense or antisense strand (siRNAs 1-6). The results of
141 All siRNAs were designed to target firefly luciferase, so we used
152 this assay are illustrated in Figure 2 and IC50 values for each
the standard Dual-Luciferase Reporter Gene Assay (Promega1) t5o3 siRNA are summarized in Table 1. The corresponding dose-
142
143 assess their gene-silencing activity. We used two expression
154 response curves are found in Supplementary Figure S-3.
144 plasmids coding for either firefly luciferase (target) or Renilla
150
5000 pM
2500 pM
1000 pM
500 pM
250 pM
50 pM
100
50
0
10 pM
5 pM
l
t
1
2
3
4
5
6
o
w
r
t
n
o
C
Figure 2. Relative normalized expression of firefly luciferase in HeLa cells after 24 hours treatment with unmodified (wt) siRNA or siRNAs bearing a single cubane
modification within either the sense or antisense strand. Firefly luciferase expression was normalized to Renilla luciferase. Error bars indicate standard deviation of
at least two independent biological replicates.
1
2
3
4
5
6
7
8
9
The resulting gene-silencing effect was position-dependent, wit2h0 Given the success of these cubane-modified siRNAs, we decided
siRNAs modified within the sense strand showing highe2r1 to investigate the effect of combining the modified strands used in
knockdown than those modified within the antisense strand. W2e2 siRNA duplexes 1-6 to yield a library of six additional siRNA
found that siRNA 1 (replacing a single dT in the sense strand 23’3 duplexes modified within both the sense and antisense strands
overhang) and siRNA 2 (modified at the sense strand position 72)4 (siRNAs 7-12). While less effective than their singly modified
showed the highest activity with IC50 values of 83.90 and 45.4205 counterparts, these doubly modified duplexes still retain good
pM, respectively. These results are comparable to other reporte2d6 biological activity with IC50 values ranging from 436.7 to 1259 pM
neutral backbone replacements in the literature.[16b] We als2o7 (Figure 3). Out of this small library, siRNA 12, which is modified
investigated the effect of replacing both dT nucleotides in th2e8 at the antisense strand 3’ end and at position 11 from the sense
10 sense strand 3’ overhang (siRNA 4) but found that this reduce2d9 strand 5’ end, displayed the highest gene-silencing efficacy (IC50:
11 siRNA activity by nearly 2 fold, with an IC50 of 152.5 pM3,0 436.7 pM). Similarly, siRNA 10, which is modified at the antisense
12 compared to the single dT replacement (siRNA 1). A similar effec3t1 strand 3’ end and at position 7 from the sense strand 5’ end,
13 was observed when we modified position 11 from the sens3e2 displayed good activity with an IC50 of 557.7 pM. On the other
14 strand 5’ end (siRNA 3; IC50: 108.8 pM). On the other hand, w3e3 hand, modifying the 3’ end of the sense strand and position 12
15 found the cubane modification was better tolerated in antisens3e4 from the antisense strand 5’ end (siRNA 7) led to a slight decrease
16 strand when placed at the 3’ end (siRNA 6; IC50: 101.9 pM) a3s5 in activity (IC50: 754.3 pM). Modifying both strands at their
17 opposed to internally (siRNA 5; IC50: 305.5 pM). Taken togethe3r,6 respective 3’ ends (siRNA 8) did not improve gene-silencing
18 these results indicate that the cubane modification is suitable fo3r7 activity (IC50: 816.0 pM). Notably, modifying position 11 from the
19 incorporation into siRNAs, in either the sense or antisense strand3.8 sense strand 5’ end and position 12 from the antisense strand 5’
39 end led to significant thermal destabilization (ΔTm: -24 °C) yet this
40 siRNA displayed effective gene-silencing activity (IC50: 573.1 pM).
3
This article is protected by copyright. All rights reserved.

10.1002/cbic.202100334
ChemBioChem
RESEARCH ARTICLE
1
2
3
4
5
On the other hand, modifying position 7 from the sense strand 5’6 gene-silencing activity, where the secondary pathway without
end and position and position 12 from the antisense strand 5’ end7 strand cleavage is activated. This increase in activity is compared
led to the lowest reported potency (IC50: 1259 pM) even though8 to
a non-cleavable spacer in the same location, where
this duplex was only destabilized by -11°C (siRNA 9). Previous9 destabilization can assist with unwinding to improve silencing
reports suggest that thermal destabilization can induce stronge1r0 activity.[17]
5000 pM
2500 pM
150
1000 pM
500 pM
250 pM
50 pM
100
50
0
l
t
7
8
9
0
1
2
o
w
1
1
1
r
t
n
o
C
Figure 3. Relative normalized expression of firefly luciferase in HeLa cells after 24 hours treatment with siRNAs bearing a single cubane modification within both
the sense and antisense strands. Firefly luciferase expression was normalized to Renilla luciferase. Error bars indicate standard deviation of at least two independent
biological replicates.
250
Antisense Target
Sense Target
200
150
100
50
0
M
8
M
0
M
0
M
0
M
M
0
M
0
M
M
0
M
0
M
M
0
M
0
M
M
0
M
0
M
M
0
M
0
M
M
0
M
0
M
d
e
p
p
p
p
p
p
p
p
p
p
p
p
p
p
p
p
p
p
p
p
p
p
p
t
8
0
0
0
0
0
0
a
.
5
0
0
5
0
0
5
0
0
5
0
0
5
0
0
5
0
0
5
0
0
e
0
r
5
0
5
0
5
0
5
0
5
0
5
0
5
0
t
5
5
5
5
5
5
5
n
U
wt
2
3
5
9
10
12
Figure 4. Relative normalized expression of firefly luciferase mRNA by the activity of either the antisense target (black) or the sense targe (gray) of unmodified (wt)
or cubane-modified siRNAs . Singly modified siRNAs (2, 3 and 5) bear the cubane modification at a single position within either the sense or antisense strand
whereas doubly modified siRNAs (9, 10 and 12) bear the cubane modification at a single position within both the sense and the antisense strand.
4
This article is protected by copyright. All rights reserved.

10.1002/cbic.202100334
ChemBioChem
RESEARCH ARTICLE
The selection of the guide strand during RISC maturation is a
crucial step for RNAi function and is dependent on factors such
as the siRNA’s 5’ sequence identity, thermodynamic stability, and
chemical modifications.[18] Ideally, the antisense strand, which is
complementary to the target mRNA, should be selected for and
become the guide strand. To further investigate the effect of our
cubane modification, we employed an RT-qPCR assay to
measure mRNA cleavage due to the incorporation of either the
antisense or sense strand within the RISC.[19] As is observed in
Fig 4, wild-type siRNA exhibits significant gene-silencing for both
antisense and sense strand targets. In order to improve and
examine the effect of cubane placement on siRNA off-target
effects, we selected three singly modified and three doubly
modified cubane siRNAs and tested them at 50, 500 and 5000
pM. As seen in Figure 4, siRNAs 2 and 3, which were modified
internally within the sense strand, retained excellent targeted
antisense gene-silencing activity, but no off-target sense strand
mediated gene-silencing. On the other hand, no significant
cleavage was observed for siRNA 5, which was modified
internally within the antisense strand. In addition, and
unsurprisingly, siRNA 9, which is modified internally within both
the sense and antisense strand, led to no target cleavage. When
the cubane modification was placed at the 3’ end of the antisense
strand (siRNAs 10 and 12), significant antisense activity is
observed despite the presence of an internally-modified sense
strand in these duplexes. Taken together, siRNAs that have the
cubane modification positioned internally within the sense strand
References
[1]
[2]
A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver,
C. C. Mello, Nature 1998, 391, 806-811.
a. C. Matranga, Y. Tomari, C. Shin, D. P. Bartel, P. D.
Zamore, Cell 2005, 123, 607-620; bA. Khvorova, A.
Reynolds, S. D. Jayasena, Cell 2003, 115, 209-216.
a) P. R. de Paula Brandão, S. S. Titze-de-Almeida, R. Titze-
de-Almeida, Mol. Diagn. Ther. 2020, 24, 61-68; b) J. Rüger,
S. Ioannou, D. Castanotto, C. A. Stein, Trends Pharmacol.
Sci. 2020, 41, 27-41.
a) M. Manoharan, Biochim. Biophys. Acta 1999, 1489, 117-
130; b) P. S. Pallan, E. M. Greene, P. A. Jicman, R. K.
Pandey, M. Manoharan, E. Rozners, M. Egli, Nucleic Acids
Res. 2011, 39, 3482-3495.
[3]
[4]
[5]
[6]
[7]
J. Wang, Z. Lu, M. G. Wientjes, J. L. Au, AAPS J. 2010, 12,
492-503.
X. Shen, D. R. Corey, Nucleic Acids Res. 2017, 46, 1584-
1600.
a) F. Eckstein, Nucleic Acid Ther. 2014, 24, 374-387; b) A.
H. Hall, J. Wan, E. E. Shaughnessy, B. Ramsay Shaw, K.
A. Alexander, Nucleic Acids Res. 2004, 32, 5991-6000.
D. Mutisya, C. Selvam, B. D. Lunstad, P. S. Pallan, A. Haas,
D. Leake, M. Egli, E. Rozners, Nucleic Acids Res. 2014, 42,
6542-6551.
a) T. C. Efthymiou, B. Peel, V. Huynh, J.-P. Desaulniers,
Bioorg. Med. Chem. Lett. 2012, 22, 5590-5594; b) T. C.
Efthymiou, V. Huynh, J. Oentoro, B. Peel, J. P. Desaulniers,
Bioorg. Med. Chem. Lett. 2012, 22, 1722-1726.
K. Tsubaki, M. L. Hammill, A. J. Varley, M. Kitamura, T.
Okauchi, J.-P. Desaulniers, ACS Med. Chem. Lett. 2020,
11, 1457-1462.
[8]
[9]
[10]
[11]
(without
a cubane modification placed internally on the
complementary antisense strand), exhibit the best strand
selection, and desired antisense targeted gene-silencing (siRNAs
2, 3, 10 and 12).
a) M. L. Hammill, C. Isaacs-Trépanier, J.-P. Desaulniers,
ChemistrySelect 2017, 2, 9810-9814; b) M. L. Hammill, G.
Islam, J.-P. Desaulniers, Org. Biomol. Chem. 2020, 18, 41-
46; c) J.P. Desaulniers, G. Hagen, J. Anderson, C. McKim,
B. Roberts, RSC Adv. 2017, 7, 3450-3454.
Conclusion
[12]
[13]
[14]
T. A. Reekie, C. M. Williams, L. M. Rendina, M. Kassiou, J.
Med. Chem. 2019, 62, 1078-1095.
P. E. Eaton, Angew. Chem., Int. Ed. Engl. 1992, 31, 1421-
1436.
In conclusion, we report the synthesis of a novel cubane
derivative phosphoramidite used to prepare a small library of
siRNAs modified at the sense strand, antisense strand, or both
strands. We then assessed the biophysical properties and
biological activity of these siRNAs. All siRNAs adopted the
desired A-form helical conformation and were able to induce gene
silencing. In addition, placing the cubane moiety within the central
region of the sense strand led to desirable increases in targeted
strand selection, thus reducing off-target effects. To the best of
our knowledge, this is the first report of an siRNA bearing a
cubane modification within its backbone. Cubane is of great
interest for medicinal chemistry applications as it can act as a
bioisostere for benzene while being less toxic and more
biologically stable. Future directions include incorporating other
cubane derivatives into the siRNA sense and antisense strands
for improving the structure-activity relationship of siRNAs.
Y.-W. Cheung, P. Röthlisberger, A. E. Mechaly, P. Weber,
F. Levi-Acobas, Y. Lo, A. W. C. Wong, A. B. Kinghorn, A.
Haouz, G. P. Savage, M. Hollenstein, J. A. Tanner, Proc.
Nat. Acad. Sci. U.S.A. 2020, 117, 16790-16798.
B. A. Chalmers, H. Xing, S. Houston, C. Clark, S.
Ghassabian, A. Kuo, B. Cao, A. Reitsma, C.-E. P. Murray,
J. E. Stok, G. M. Boyle, C. J. Pierce, S. W. Littler, D. A.
Winkler, P. V. Bernhardt, C. Pasay, J. J. De Voss, J.
McCarthy, P. G. Parsons, G. H. Walter, M. T. Smith, H. M.
Cooper, S. K. Nilsson, J. Tsanaktsidis, G. P. Savage, C. M.
Williams, Angew. Chem. Int. Ed. 2016, 55, 3580-3585.
a) M. J. Falkiner, S. W. Littler, K. J. McRae, G. P. Savage,
J. Tsanaktsidis, Org. Process Res. Dev. 2013, 17, 1503-
1509; b) M. Bliese, J. Tsanaktsidis, Aust. J. Chem. 1997,
50, 189-192.
[15]
[16]
[17]
H. Addepalli, Meena, C. G. Peng, G. Wang, Y. Fan, K.
Charisse, K. N. Jayaprakash, K. G. Rajeev, R. K. Pandey,
G. Lavine, L. Zhang, K. Jahn-Hofmann, P. Hadwiger, M.
Manoharan, M. A. Maier, Nucleic Acids Res. 2010, 38,
7320-7331.
Acknowledgements
[18]
[19]
a) J. Lisowiec-Wąchnicka, N. Bartyś, A. Pasternak, Sci.
Rep. 2019, 9, 2477; b) C. L. Noland, J. A. Doudna, RNA
2013, 19, 639-648; c) A. J. Varley, J.-P. Desaulniers, RSC
Adv. 2021, 11, 2415-2426.
A. Varley, M. L. Hammill, L. Salim, J.-P. Desaulniers,
Nucleic Acid Ther. 2020, 30, 229-236.
The authors thank Ifrodet Giorgees for performing mass
spectrometry analysis and the Natural Sciences and Engineering
Research Council (NSERC) for funding.
Keywords: cubane • siRNA • gene-silencing• strand selection •
phosphoramidite
5
This article is protected by copyright. All rights reserved.

10.1002/cbic.202100334
ChemBioChem
RESEARCH ARTICLE
Entry for the Table of Contents
siRNA lego: cubane-functionalized siRNAs lead to potent RNAi-mediated gene-silencing activity, and improved strand selection.
Institute and/or researcher Twitter usernames: @JP_Desaulniers @DesaulniersLab @ontariotech_u
6
This article is protected by copyright. All rights reserved.