4′-Guanidinium-modified siRNA: A molecular tool to control RNAi activity through RISC priming and selective antisense strand loading

Article abstract of DOI:10.1039/c9cc04141a

We designed novel 4′-C-guanidinocarbohydrazidomethyl-5-methyl uridine (GMU) modified small interfering RNA (siRNA) and evaluated its biophysical and biochemical properties. Incorporation of GMU units significantly increased the thermodynamic stability as

Full text of DOI:10.1039/c9cc04141a

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DOI: 10.1039/C9CC04141A
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4′-Guanidinium-modified siRNA: A molecular tool to control RNAi
activity through RISC priming and selective antisense strand
loading †
Received 00th January 20xx,
Accepted 00th January 20xx
Ganesh N. Nawale,a Saeed Bahadorikhalili,a Pallabi Sengupta,b Sandeep Kadekar,a Subhrangsu
DOI: 10.1039/x0xx00000x
Chatterjee,b and Oommen P. Varghesea,*
www.rsc.org/
We designed novel 4′-C-guanidinocarbohydrazidomethyl-5- improve
immunocompatibility.17
To
develop
GMU
methyl uridine (GMU) modified small interfering RNA (siRNA) modification, we first synthesized guanidinocarbohydrazide
and evaluated their biophysical and biochemical properties. ligand (3) by modifying our recently reported method (Scheme
Incorporation of GMU units significantly increased 1).18
thermodynamic stability as well as the enzymatic stability
against nucleases in human serum. Gene silencing
experiment indicated that GMU modfied siRNA (siRNA6)
resulted in ≈4.9-folds more efficient knockdown than
unmodified siRNA.
Since the discovery of synthetic siRNA as a potent gene-
silencing molecule in mammalian cells, siRNA-based drugs
have emerged as a promising technology for the treatment of
a variety of diseases.1 The RNA interference (RNAi) is an
endogenous pathway for post-transcriptional gene silencing
Scheme 1. Synthesis of guanidinocarbohydrazide ligand (3) and 4′-C-
that uses sequence-specific knockdown of target mRNA. In this
mechanism, short double-stranded RNA (21–23-mer)
undergoes a natural antisense or guide strand selection
process into the RNA-induced silencing complex (RISC) in the
cytosol.2-4 This activated RISC binds mRNA, resulting in gene
guanidinicarbohydrazidomethyl-5-methyl uridine phosphoramidite (12). Reagents and
conditions: i) S-methylisothiourea hemisulfate, DCM/sat. NaHCO3, 45 °C, h; ii)
carbohydrazide, MeOH, reflux, 12 h; iii) NaOMe/MeOH, room temperature (rt), 3 h; iv)
TBDMS-Cl, imidazole, DMF, rt, 2 h; v) 1 M DIBAL/hex, DCM, -78 °C, 2 h; vi) Compound 3,
MeOH, reflux, 5 h; vii) NaBH4, MeOH, rt, 3 h; viii) EtOCOCF3, THF, rt, 4 h; ix) 20 %
Pd(OH)2/C, ammonium formate, EtOH, 70 °C, 48 h; x) DMTr-Cl, Py, DMAP, 45 °C, 3 h; xi)
2
silencing.5, 6 Even though the RNAi based drug development DIPEA, CEP-Cl, DCM, rt, 3 h.
platform looks promising, there are several challenges to
The S-methylisothiourea hemisulfate and activated
overcome. These include challenges to enhance the stability of
such drugs against nucleases,7 minimize off-target effects,8
immune activation,9 and most importantly to enhance in vivo
delivery.10 We have recently developed the first non-cationic
non-viral siRNA delivery method using hyaluronic acid, a
natural biopolymer present in the extracellular matrix.11
In this communication, we present synthesis of a novel 4′-
guanidinium modified siRNA and their systematic
physicochemical, biochemical, computational and in vitro gene
silencing analysis. The GMU modification at the 3′-end is
expected to be protonated at the physiological condition,12, 13
which could enhance nuclease stability,14-16 and potentially
hydroxypropionitrile compound 1 was reacted to obtain
compound 2. The S-methyl group was further substituted with
carbohydrazide to furnish guanidinocarbohydrazide ligand 3.
To prepare the GMU building block, we first synthesized the
cyano nucleoside 4 from D-glucose using our previously
reported procedure.19 The 2′-acetate group of nucleoside 4
was hydrolysed using sodium methoxide to yield 2′-hydroxy
nucleoside 5. Compound 5 was further silylated using tert-
butyldimethylsilyl chloride (TBDMS-Cl) to obtain 2′-O-silyl (TBS)
nucleoside 6.20 We further reduced the cyano group of 6 to
obtain crude aldehyde modified nucleoside 7a which was
subsequently converted to hydrazone nucleoside 7.21 The
hydrazone derivative was further reduced to afford guanidino
nucleoside 8. We further protected the amino group of the
hydrazine moiety of 8 using ethyltrifluoro acetate (CF3COOEt)
to obtain trifluoro acetate (TFA) protected nucleoside 9. The
debenzylation of 9 was performed using 20 % Pd(OH)2 to
a. Translational Chemical Biology Laboratory, Division of Polymer Chemistry,
Department of Chemistry-Ångström, Uppsala University, Uppsala, Sweden
b. Biomolecular NMR and Drug Design Laboratory, Department of Biophysics, Bose
Institute, P-1/12 CIT Scheme VII M, Kolkata, India.
† Electronic Supplementary Information (ESI) available: Experimental details,
spectroscopic and analytical data. See DOI: 10.1039/x0xx00000x
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obtain the deprotected diol nucleoside 10. The 5′ hydroxyl of performing MM-PBSA calculations, which decomposes the
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compound 10 was selectively protected using 4,4′- interaction energies for the modified reDsiOdIu: 1e0s.10b3y9/cCo9nCsCid0e41r4in1Ag
dimethoxytrityl chloride (DMTr-Cl) to furnish 11.22 Finally, molecular mechanics and solvation energies (Table S1).
phosphitylation of compound 11 was carried out to furnish the
phosphoramidite 12.22 The unmodified and GMU modified
siRNAs targeting signal transducer and activator of
transcription 3 (STAT3) mRNA were synthesized using a solid
phase RNA synthesizer (Table 1). The cleavage from the solid
support and deprotection of guanidinium and phosphate
protecting groups were carried out using 50 % piperidine.23
Further nucleobase deprotection, 2′-O-TBDMS group
deprotection was carried out and the product was purified by
denaturing polyacrylamide gel electrophoresis (PAGE).22 In
order to evaluate the effect of chemical modification on the
thermal stability of siRNA duplexes, we performed UV-melting
studies (Table 1).
Fig. 1. The RNAi activity of unmodified and GMU modified siRNAs. (A) Time-
dependent RNAi activity after 24 h, 48 h and 96 h of transfection with 1 nM
Table 1. The sequences of siRNA duplexes and Tm values
siRNA. (B) Concentration-dependent RNAi activity post 96 h of transfection.
b
Passenger (5′-3′, above) and guide
strand (3′-5′, below)
∆Tm
/
a
Name
Tm
Experiments were performed in triplicate for three times. The Student's t-test
was used to determine statistical differences between pairs of groups (* = p ≤
0.05).
mod.
GGAAGCUGCAGAAAGAUACTT
TTCCUUCGACGUCUUUCUAUG
GGAAGCUGCAGAAAGAUACTT
TTCCUUCGACGUCUUUCUAUG
GGAAGCUGCAGAAAGAUACTT
TTCCUUCGACGUCUUUCUAUG
GGAAGCUGCAGAAAGAUACTT
TTCCUUCGACGUCUUUCUAUG
GGAAGCUGCAGAAAGAUACTT
TTCCUUCGACGUCUUUCUAUG
GGAAGCUGCAGAAAGAUACTT
TTCCUUCGACGUCUUUCUAUG
siRNA1
siRNA2
siRNA3
siRNA4
siRNA5
siRNA6
66.9 ± 0.1
67.8 ± 0.3
69.8 ± 0.1
69.5 ± 0.2
69.7 ± 0.3
70.3 ± 0.2
+0.9
+2.9
+2.6
+2.8
+3.4
The modified siRNAs were further evaluated for their RNAi
efficacy in human colon cancer cell line (HCT116). HCT116 cells
were transfected with different concentrations of siRNA, and
the expression levels of STAT3 mRNA was analyzed after 24 h,
48 h or 96 h (Fig. 1). The scrambled siRNA sequence (SCR) was
used as a negative control in these experiments. The gene
knockdown (KD) results of unmodified and GMU modified
siRNAs are summarized in Fig. 1. These results indicate that
the modifications of overhang are well tolerated in passenger
strand as well as guide strand of siRNA (Fig. 1A). The
modifications in passenger strand of siRNA duplexes such as
siRNA2 and siRNA3 have shown no loss of RNAi activity when
compared to the unmodified siRNA1 (in 48 or 96 h 2-5 % less
KD). The effect of modifications in the guide strand (siRNA4),
however, was found to be sensitive with 20-40 % less KD
efficiency as compared to siRNA1. Surprisingly, when both the
strand of siRNA were modified in an asymmetric manner with
single modification on passenger and two modifications on
guide strand (siRNA5), there was a complete loss of RNAi
activity. Interestingly, in the case of siRNA6 where both the
overhangs were modified with GMU in a symmetrical manner,
the RNAi activity was restored and was in fact was superior (7
% more KD for 96 h) to the unmodified siRNA (Fig. 1A). We
further performed concentration-dependent study to validate
the actual difference in RNAi activity between siRNA1 and
siRNA6. Gratifyingly, these experiments suggested that siRNA6
with completely modified overhangs were ≈4.9 folds more
efficient as compared to the siRNA1 control (IC50 for siRNA1
≈311 pM, while for siRNA6 ≈63 pM, Fig. S3). Specifically, the
siRNA6 demonstrated 20–40 % higher activity as compared to
the native siRNA after 96 h (Fig. 1B). One of the key aspects of
siRNA duplex that dictates its activity is RISC priming, which
enable correct orientation of the duplex for specific loading of
the antisense strand.24 The 3′-overhang plays an important
role in this step as it binds to the PAZ domain of the
aTm represents melting temperatures for unmodified and GMU modified
siRNA duplexes (red colour indicates modification) in °C. b∆Tm represents
the [Tm (RNA mod.) − Tm (RNA unmod.)]. The Tm values were determined
using 1 µM of siRNA in buffer containing 50 mM NaCl, 10 mM Na2PO4, pH
7.4. All experiments were triplicated, and the Tm values have reported an
average of 3 measurements with the estimated standard deviation.
Incorporation of single GMU modification at the 3′-terminal of
the passenger strand of siRNA (siRNA2) showed a change in
melting temperature (∆Tm) of +0.9 °C per modification as
compared to the unmodified duplex. Incorporation of GMU
modification on both the overhang nucleotides of siRNA in
either passenger (siRNA3) or guide (siRNA4) strand
demonstrated a similar increase in the melting temperature
(∆Tm = 2.6–2.9 °C). When both the strands of siRNA were
modified together, an increase in ∆Tm of 2.8–3.4 °C was
observed (siRNA5 and siRNA6). In general, GMU modifications
resulted in a significant thermal stabilization of all the modified
siRNA duplexes (Table 1).
To elucidate the role of guanidinium modifications in
thermal stability of siRNA duplexes, we performed in silico
molecular modelling and simulation of the modified and
unmodified siRNA duplexes (Fig. S1-S2). These studies implied
that the guanidinium linkers near 3′- ribonucleotides (C4′ of
ribose sugar) and penultimate thymidine residues
(deoxyribose sugar) at siRNA6 termini conferred additional
non-covalent interactions to protect the fraying ends of siRNA
duplex and impart thermodynamic stability. In siRNA6, dual
GMU modifications at 3′-overhang amplified the network of
electrostatic interactions with adjacent residues (Fig. S1B). We
further analyzed the energetics of siRNA conformers, by
2 | J. Name., 2012, 00, 1-3
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Argonaute-2 protein within the RISC.25 Intrigued by the that the sugar puckering of siRNA6 remained inVCie3w′-AertnicdleoOn(lNine-
difference in bioactivity between siRNA5 and siRNA6, we were type sugar) sugar conformation (Fig. S5)D, OwI:h1i0c.h10i3s9/eCs9sCeCn0ti4a1l4t1oA
prompted to investigate the differences in actual loading of maintain the integrity of A-type RNA.
sense and antisense strands. For this purpose, we performed
One of the major challenges of the nucleic acid drug is the
recruitment assay using stem-loop qPCR.26 This experiment stability towards endo and exonucleases present in blood
clearly indicated enhanced recruitment of guide strand in case plasma and within the cytosol.7 We have previously shown
of modified siRNA6 (≈1.8-fold enhancement as compared to that a single guanidinium modified locked nucleoside at the 3′-
the siRNA1; Fig. 2A) while the recruitment of guide strand in terminal can impart significant enzymatic stability for over 24 h
siRNA5 was not observed (Fig. 2A). Interestingly, we observed in human serum.18 This prompted us to evaluate the stability
exclusive recruitment of sense strand in case of siRNA5 (≈234- of the GMU modified siRNAs in human serum. Incubation of
fold enhancement as compared to the siRNA1) and modified and unmodified siRNAs with 70 % human serum at
significantly less in siRNA6 (0.5 fold less compared to the various time points followed by PAGE analysis of the nuclease
siRNA1 (Fig. 2B).
digested product indicated that all modified sequences
Fig. 2. Strand recruitment and RISC binding of GMU modified siRNA. (A) Effect of (siRNA2-6) showed a significant improvement in nuclease
stability (Fig. 3A).
Fig. 3. Serum stability for modified siRNA. (A) 15 % Native PAGE denoting
modification on guide (AS) strand recruitment, and (B) on passenger (SS) strand
recruitment within RISC as analyzed by stem-loop qPCR with the scrambled
sequence as a control. (C) Recruitment of siRNA6 into the PAZ domain of
Argonaute protein. (D) Principal non-covalent intermolecular interactions
stability of siRNA duplexes (30 µM) after incubating in 70 % human serum (t = 0,
between the PAZ domain of Argonaute protein and siRNA6. Experiments were
1, 2, 4 and 8 hours). h* indicates serum stability study for up to 72 h for siRNA6.
performed in triplicate for three times. The Student's t-test was used to
The intensity of the full-length siRNA (intact overhang) bands decreases with an
determine statistical differences between pairs of groups (* = p ≤ 0.05, ** = p ≤
increase in incubation time. (B) RNase A-siRNA1 complex. (C) The catalytic core
0.01 and *** = p ≤ 0.001).
of RNase A (His119, Cys41, and His12) providing in-line attack in a siRNA1
duplex. (D) RNase A-siRNA6 complex. (E) The catalytic core (His119, Cys41, and
To further investigate the role of guanidinium modifications in
guide strand recruitment, we carried out molecular docking
studies between Argonaute protein and siRNA duplexes
His12) of RNase A is spatially placed far apart from siRNA6 to invade the double-
stranded RNA.
(siRNA1 and siRNA6) and compared the intermolecular
The siRNA duplex with single or double GMU modifications at
the 3′-end of the passenger or guide strand showed stability
for over 4 h in human serum. The dual modification on both
guide and passenger strands (siRNA6) further enhanced
nuclease stability with nearly intact duplex after 12 h and
almost 50 % intact siRNA after 72 h (h*, Fig. 3A). The
unmodified siRNA duplex (siRNA1) on the other hand was
completely degraded within one hour of incubation time. We
reasoned that the stabilized non-covalent interactions by GMU
modifications at 3′-termini sequester siRNA molecules from
the catalytic pockets of RNase A-type endonucleases, which
enhance nuclease resistance of the siRNA molecules in the
sera. Structural analyses of in silico RNase A-siRNA complexes
further underscored that RNase A catalytic triad (His12,
His119, and Lys41) invades siRNA1 to promote RNA cleavage
(Fig. 3B and 3C). However, the Watson-Crick pairing in siRNA6
is perturbed due to the end modifications rendering reduced
efficiency of RNase A catalytic triad to remain in the spatial
proximity of the ribonucleotides, required for the nucleophilic
attack towards 2′-OH of the ribose sugars. Therefore, siRNA6 is
interactions. The PAZ domain of Argonaute, which is regarded
as the principal RNA binding module to recognize the 3′
overhang of siRNA is observed to make stable non-covalent
interactions with siRNA6, consistent with our experimental
evidence (Fig. 2C). Symmetric guanidinium modifications at
both ends favoured spatial positioning of siRNA into the
binding pocket of PAZ domain of RISC complex. Thr 361, Ile-
353, Lys-355 allow the passage of siRNA1 and siRNA6 into RISC
complex (Fig. S4B and 3D). In siRNA6, Cys-352 and Thr-368
additionally play important roles to constitute electrostatic
interactions with A10, G8 and U7 and enable improved strand
recruitment into RISC complex (Fig. 2D). Structural dynamics of
siRNA1 evidenced stable Watson-Crick base pairing in the
central domain, which spanned the binding pocket of PAZ. We
observed strong pairing between G11:C9, stabilized by
neighbouring G8, which projected a π–π stacking interaction
with C9 in the same strand. This sequestered the hydrogen-
bond interaction between Lys 355 and C9, which might play a
critical role to compromise its recruitment into RISC complex
(Fig. S4A and S4B). Further structural investigations confirmed
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15 M. Kanazaki, Y. Ueno, S. Shuto and A. Matsuda, J. Am. Chem.
not as vulnerable as siRNA1 and is able to evade nuclease
cleavage (Fig. 3D and 3E).
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Soc., 2000, 122, 2422-2432.
DOI: 10.1039/C9CC04141A
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In summary, we have synthesized novel 4′-guanidinium-
modified amidite to obtain several chemically modified siRNAs.
These modified siRNAs not only increase thermal stability but
also significantly improved stability in human serum. The
incorporation of GMU nucleotides at the two terminal 3′-
overhangs (siRNA6) also resulted in sustained gene silencing
activity at significantly lower concentration (picomolar) after
96 h of transfection. The stem-loop qPCR experiments
confirmed that such sustained activity was due to enhanced
guide strand recruitment within the RISC complex. Structural
investigations of the Argonaute protein and siRNA6 also
confirmed altered spatial positioning of siRNA into PAZ domain
resulting in successful recruitment of the desired guide strand
into RISC complex and enhanced activity. We believe that the
4′-guanidino modifications at the 3′-overhangs of siRNA will
allow development of next generation of RNAi drugs with
enhanced potency and minimal sense-strand mediated off-
target effects. Since such modifications are incorporated at the
overhang region, it could be adapted to other siRNA
sequences, without losing its bioactivity.
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Conflicts of interest
There are no conflicts to declare.
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