Synthesis and Biological Activity of Short Interfering RNAs Having Several Consecutive Amide Internucleoside Linkages

Article abstract of DOI:10.1002/chem.201903754

The success of RNA interference (RNAi) as a research tool and potential therapeutic approach has reinvigorated interest in chemical modifications of RNA. Replacement of the negatively charged phosphates with neutral amides may be expected to improve bioav

Full text of DOI:10.1002/chem.201903754

A Journal of
Accepted Article
Title: Synthesis and Biological Activity of Short Interfering RNAs
Having Several Consecutive Amide Internucleoside Linkages
Authors: Venubabu Kotikam, Julien A Viel, and Eriks Rozners
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Synthesis and Biological Activity of Short Interfering RNAs
Having Several Consecutive Amide Internucleoside Linkages
Venubabu Kotikam,*[a] Julien A. Viel[a] and Eriks Rozners*[a]
Dedication ((optional))
Abstract: The success of RNA interference (RNAi) as a research tool
and potential therapeutic approach has reinvigorated interest in
chemical modifications of RNA. Replacement of the negatively
charged phosphates with neutral amides may be expected to improve
bioavailability and cellular uptake of small interfering RNAs (siRNAs)
critical for in vivo applications. In this study, we introduced up to seven
consecutive amide linkages at the 3´-end of the guide strand of an
siRNA duplex. Modified guide strands having four consecutive amide
linkages retained high RNAi activity when paired with a passenger
strand having one amide modification between its first and second
nucleosides at the 5´-end. Further increase in the number of
modifications decreased the RNAi activity; however, siRNAs with six
and seven amide linkages still showed useful target silencing. While
an siRNA duplex having nine amide linkages retained some silencing
activity, the partial reduction of the negative charge did not enable
passive uptake in HeLa cells. Our results suggest that further
chemical modifications, in addition to amide linkages, are needed to
its own mRNA targets to be cleaved, and resulting in off-target
effects. The success of in vivo applications of RNAi, including
clinical trials, has been limited by the siRNA-associated issues of
challenging systemic delivery, inadequate cellular uptake, and off-
target microRNA-like activity of both guide and passenger
strands.[2a]
The large size and negatively charged phosphate backbone of
siRNAs cause the problems of poor bioavailability, short half-life
in plasma, and poor cellular uptake. Complexation with lipids and
liposomes improves bioavailability and cellular uptake[5] and
enables in vivo delivery of siRNAs.[6] However, lipid nanoparticles
are not an ideal solution due to limited diffusion and poor
pharmacokinetics caused by their large size (~100 nm and 100
megaD). Conjugation with spermine[7] and cholesterol[8] has also
been used to deliver siRNAs in cultured cells and mice.
Conjugation with N-acetylgalactosamine (GalNac) enables
efficient delivery of siRNAs to hepatocytes via the
enable cellular uptake of siRNAs in the absence of transfection agents. asialoglycoprotein receptor,[5b] and achieves 80% reduction of
transthyretin, a protein implicated in amyloidosis, in non-human
primates.[9] Chemical modifications of RNA sugar-phosphate
backbone have also been used extensively to improve the
properties of siRNAs.[10] Patisiran, the recently approved first
Introduction
RNAi drug, combines strategically-placed chemical modifications
11]
with liposome formulation for delivery.[2d,
Despite these
RNA interference (RNAi) has become a well-established and
powerful tool in the broad fields of biochemistry and molecular
biology,[1] and has an intriguing potential to become a new
therapeutic approach, with more than 20 RNAi-based clinical
trials currently being pursued.[2] Natural RNAi is driven by external
(invading virus) or endogenous (microRNA) double-stranded
RNAs (dsRNA) that are processed by cellular endonucleases
(Drosha and Dicer) into 21-23 nucleotide-long short interfering
RNAs (siRNAs) that are loaded into Argonaute 2 (Ago2), the
principal protein of RNAi.[3] For research or therapeutic
applications, the initial endonuclease-processing step may be
bypassed by directly delivering synthetic siRNAs.[4] During
loading, an siRNA duplex is unwound and its guide strand
(complimentary to the target mRNA) is incorporated into Ago2.
The loaded guide strand directs Ago2 to cleave complementary
target-mRNA, preventing the mRNA from being translated into
protein, thus silencing the target gene. Ideally, only the guide
strand of an siRNA would be loaded into Ago2. However, in real
systems, the passenger strand may be loaded instead, causing
promising results, current systemic delivery is limited mostly to the
liver and spleen. Off target effects, poor biodistribution, and poor
cellular uptake remain bottlenecks for the broader in vivo
application of siRNAs.
We envisioned that the replacement of phosphates with neutral
backbone modifications, such as amide linkages, might
complement currently-used modifications and provide additional
improvements of bioavailability and cellular uptake of siRNAs.
Our hypothesis was inspired by the recent work of Dowdy and co-
workers,[12] who showed that bioreversible phosphate
neutralization by phosphotriesters enabled efficient siRNA
delivery after conjugation with cell-penetrating peptides. In the
present study, we tested the limits of consecutive amide linkages
that could be placed in an siRNA duplex while retaining useful
RNAi activity. Earlier, we showed[13] that the replacement of
isolated phosphates with amide linkages did not change the
thermal stability and overall A-type conformation of short RNA
duplexes. In more recent studies,[14] we discovered that amides
were well tolerated at most positions of siRNA guide strands,
except between the first and second nucleosides, and actually
increased the RNAi activity when placed in the middle of a guide
strand. Most remarkably, an amide linkage between the first and
second nucleosides of the passenger strand completely
abolished its undesired activity and increased the desired activity
of the complementary guide strand.[14b] Taken together, these
a]
Dr. V. Kotikam, J. A. Viel, Prof. Dr. E. Rozners
Department of Chemistry, Binghamton University
Binghamton, New York 13902, USA
E-mail: erozners@binghamton.edu or vkotikam@binghamton.edu
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/chem.201903754
Chemistry - A European Journal
FULL PAPER
5' HO
results suggested that amides were promising modifications for
the optimization of siRNAs, and prompted us to explore the limits
of amide modification in an siRNA duplex.
MMTrO
O
Base
Base
O
P
O
O
O
O
O
O
OH
OTBS
1a Base= Ura
1b Base= AdeBz
Base
O
HN
OH
Base
O
Herein we report that up to five amide linkages were well tolerated
in an siRNA duplex. An siRNA having four consecutive amide
linkages in the guide strand maintained high silencing activity
(~80% at 10 nM) when the passenger strand had an amide
linkage between its first and second nucleosides. A similar result
was obtained for an siRNA having three and two consecutive
amide linkages in the guide and passenger strands, respectively.
Further increases of amide modifications in either the guide or
passenger strand decreased RNAi activity; however, siRNAs with
six and seven amide linkages still showed useful silencing of
target gene expression. An siRNA duplex with nine amide
linkages did not show any notable RNAi activity in the absence of
lipid transfection agent, suggesting that further modification is
needed to explore if reducing charge may have a beneficial effect
on the cellular uptake of siRNAs.
OH
O
MMTrHN
O
O
P
O
Base
O
OH
O
Base
O
HN
Base
O
OTBS
O
O
OH
2a Base= Ura
2b Base= AdeBz
OH
OH
O
O
P
HN
Base
O
O
O
O
Base
MMTrHN
OH
MMTrHN
Ura
Ura
O
O
OH
O
O
3
HN
OH
O
Base
O
OTOM
TOMO
O
O
O
NC
P
N
O
P
O
4
O
O
3'
OH OH
Figure 1. Structures of 3´-end and 5´-end amide-modified RNAs and the
building blocks required for their preparation.
Table 1. Sequences of on-target siRNA (G0-P0), amide-modified passenger
and guide strands, and non-target control siRNA (NTC) used in the RNAi
experiments.
Results and Discussion
Overall design. In an earlier preliminary study,[15] we used uridine
amino acid 2a (Figure 1) along with chemistry-switching (switch)
monomers 1a and 4 to incorporate short stretches of amide-linked
uridines in the middle of a short RNA duplex and near the 5´-end
of the passenger strand of an siRNA. We found that three
consecutive amides did not interfere with normal base-pairing and
A-type conformation of dsRNA, and caused little change in RNAi
activity.[15] In the present study, we tested how many amide
linkages can be introduced at the 3´-end of the guide strand and
5´-end of the passenger strand using both modified uridine and
modified adenosine nucleosides (Figure 1). Our goal was to
create an siRNA duplex having a stretch of non-ionic backbone at
one of the ends. Previous studies by Iwase et al.[16] showed that
siRNAs having two consecutive amide linkages at the 3´-ends of
both guide and passenger strands maintained full RNAi activity.
In the present study, we started with three and introduced up to
seven consecutive amides at the 3´-end of the guide strand. The
passenger strand had either a single amide or two consecutive
amides at its 5’-end. The synthesis of 3´-amide-modified guide
strands (G3-G7, Table 1) started with attachment of the first
monomer, 5´-amino 2´-succinyluridine 3 (Figure 1), to the solid
support. This was followed by peptide-like couplings of uridine 2a
and adenosine 2b amino acids to assemble the amide-linked RNA
portion. Amide-to-phosphate switch monomer 1a or 1b was then
introduced, allowing for the syntheses of G3-G7 to be completed
using standard RNA 2´-O-TOM phosphoramidites (Glen
Research). Preparation of the passenger strand having two 5’-
amide-modifications (P2, Table 1) started with standard
automated RNA synthesis. Phosphate-to-amide switch monomer
4 was then introduced, allowing for assembly of the amide-linked
RNA portion using the adenosine 2b and uridine 2a amino acids.
Main Text Paragraph.
Code[a]
N[b]
RNA sequence[c]
3´-UU GUU AAG CAU CCA GUU UUA U-5´ (passenger)
5´-CAA UUC GUA GGU CAA AAU AUU-3´ (guide)
G0-P0
0
P0
P1
P2
G0
G3
G4
G5
G6
G7
0
1
2
0
3
4
5
6
7
3´-UUG UUA AGC AUC CAG UUU UAU-5´
3´-UUG UUA AGC AUC CAG UUU UAamU-5´
3´-UUG UUA AGC AUC CAG UUU UamAamU-5´
5´-CAA UUC GUA GGU CAA AAU AUU-3´
5´-pCAA UUC GUA GGU CAA AAUamAamUamU-3´
5´-pCAA UUC GUA GGU CAA AAamUamAamUamU-3´
5´-pCAA UUC GUA GGU CAA AamAamUamAamUamU-3´
5´-pCAAUUCGUAGGU CAAamAamAamUamAamUamU-3´
5´-pCAAUUCGUAGGU CAamAamAamAamUamAamUamU-3´
3´-UU AUC GCU GAU UUG UGU AGU U-5´ (passenger)
5´-UAG CGA CUA AAC ACA UCA A UU-3´ (guide)
NTC
0
[a] P and G denote the passenger and guide strands, respectively; NTC
denotes non-target control siRNA. [b] N is number of amide modifications.
[c] In RNA sequences, p denotes a phosphate monoester at the 5´-end and
each am denotes an internucleoside amide linkage. U or A denotes either
a phosphate-to-amide (4) or amide-to-phosphate (1a or 1b) switch monomer
being used during solid phase synthesis.
Synthesis of monomeric building blocks. The key monomers
for preparation of amide-modified RNA were the nucleoside
amino acids 2a and 2b (boxed in Figure 1). In earlier work,[15] we
synthesized the uridine monomer 2a starting from D-xylose.[17]
However, such a route required 18 steps and gave 2a in only 4%
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10.1002/chem.201903754
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overall yield. Moreover, we were unable to make adenosine
monomer 2b following this route because of elimination of the
adenine heterocycle during attempts to introduce the 2´-O-TBS
protection. In the present study, we used our recently developed
and more efficient synthesis route that gave 2a in only six steps
and 31% overall yield and 2b in eight steps and 16% overall
yield.[18] Switch monomers 1a and 1b were prepared as previously
reported.[14a] The synthesis of monomers 3 and 4 (for details, see
sequences were synthesized by standard phosphoramidite
chemistry using 2´-O-TOM protected monomers (Glen Research)
on an Expedite 8909 DNA/RNA synthesizer. The amide-modified
guide and passenger strands were cleaved from the solid support,
deprotected, purified, and analyzed (MALDI-TOF MS) using
standard RNA synthesis protocols, as recommended by Glen
15]
Research and previously reported by us.[14a,
Overall, the
modest amide coupling yields enabled synthesis of sufficient
amounts of modified RNA strands having up to six consecutive
amide linkages (for yields, see Table S1 and Figures S1-S12). We
obtained only a small amount of G7 (0.11 nmols).
Supporting Information) started with
a non-selective TOM
protection of 5´-azidouridine (Scheme 1) that gave a separable
mixture of 3´- and 2´-O-TOM isomers 5 and 6, respectively.
Reduction of the azide was followed by 5´-tritylation to give 7 and
8, which were converted into succinylated monomer 3 and switch
monomer 4 using standard nucleoside chemistry procedures.
RNAi activity of siRNAs having consecutive amide linkages.
The effect of amide modifications on RNAi silencing of the target,
PPIB, was studied in HeLa cells (CCL2 variant) using a bDNA
assay (QuantiGene 2.0 Reagent System, Figure S13) with GAPD
(glyceraldehyde-3-phosphate dehydrogenase) serving as an
internal reference. Initial experiments showed that RNAi activities
of amide-modified guides G3 and G4, each paired with the
unmodified passenger P0, were both <20% at 1 nM duplex
concentration. For comparison, unmodified guide G0 yielded an
activity of ~85% under the same conditions. Increasing siRNA
duplex concentrations to 100 nM somewhat improved the
silencing activity to 35%, 25%, and 90% for G3, G4, and G0,
respectively (Figure S14). This result was consistent with our
earlier study[14a] that showed that isolated amide linkages at most
internal positions of this guide strand strongly reduce its RNAi
activity. We later showed[14b] that the decreased silencing activity
was due to preferential loading of the unmodified passenger P0
(instead of the modified guides) into Ago 2, and that the activity
could be restored by using passenger strand P1, having a single
amide linkage between its first and second nucleosides.
Consistent with these earlier findings, pairing either G3 or G4 with
P1 resulted in >80% silencing activity at both 100 and at 10 nm
duplex concentrations (Figure 2).
N3
MMTrHN
(b,c)
MMTrHN
Ura
Ura
Ura
O
O
O
O
3
(d)
OH
N3
TOMO
N3
OH
TOMO
OH
TOMO
O
Ura
O
(a)
5
7
O
MMTrHN
OH OH
5'-azido-5'-dexoyuridine
MMTrHN
(b,c)
Ura
Ura
Ura
O
(e)
O
O
O
OTOM
OH OTOM
6
OH OTOM
8
O
NC
P
N
4
Scheme 1. Synthesis of monomers 3 and 4. Steps: (a) TOM-Cl, Bu2SnCl2,
DIPEA, ClCH2CH2Cl, reflux, 1 h, 26% of 5 and 41% of 6; (b) H2S (gas),
pyridine:water (4:1), rt, overnight; (c) p-methoxytrityl chloride, DMAP, pyridine,
rt, overnight, 65% of 7 and 65% of 8; (d) succinic anhydride, DBU, CH2Cl2, rt,
30 min, 80%; (e) P(OCH2CH2CN)(N(iPr)2)2, tetrazole, N-methylimidazole,
CH2Cl2, rt, 24 h, 35%.
Design and synthesis of siRNAs containing consecutive
amide linkages. For the current study, we chose an siRNA (G0-
P0, Table 1) that targets the mRNA of cyclophilin
B (a
housekeeping gene, peptidylprolyl isomerase B, PPIB) which we
have previously used[14] to study the effect of amide modifications
in RNAi. The G0-P0 duplex was rich in Us and As at the 3´-end of
the guide and 5´-end of the passenger strand, which made
synthesis of amide-linked RNAs easier because only modified U
14b]
and A monomers were needed. Our previous studies[14a,
showed that both guide and passenger strands of this siRNA were
highly active (the passenger was also loaded in Ago2) and that
isolated amide modifications in the guide strand strongly reduced
its activity when paired with an unmodified passenger strand.
Hence, G0-P0 was a good model system due to its relatively easy
synthesis and high sensitivity to amide modification, which would
provide a rigorous test of the limits of consecutive amide
modifications that may be tolerated in an siRNA.
Synthesis of amide-modified guide strands G3-G7 was done on
long chain alkylamine controlled pore glass (CPG, standard solid
support for DNA/RNA synthesis, Glen Research) derivatized with
monomer 3 through its succinamide linker (for details, see
Supporting Information). Synthesis of amide-modified passenger
strand P2 was performed on standard U-derivatized CPG RNA
support (Glen Research). Amide couplings were done using
either PyAOP (for G3-G7) or HATU (for P2) as activators for
carboxylic acid monomers 2a and 2b (Figure 1); coupling yields
were in the 80-85% range. The unmodified RNA parts of all
Figure 2. Concentration-dependent silencing activity of amide-modified guide
strands G3, G4, G5, and G6 (for sequences, see Table 1) paired with passenger
strand P1 having
a single amide linkage between its first and second
nucleosides. Activity is presented as a PPIB-to-GAPD mRNA ratio in HeLa
(CCL2) cells, normalized to a non-target control (NTC). G0-P1 is the positive
control. The data are averages of biological duplicate experiments, each
performed as a technical triplicate.
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Overall, all amide-modified guide strands (G3 through G6)
showed the expected concentration-dependent silencing of the
target, PPIB. While the activity of G3-P1 was similar to that of the
positive control G0-P1 at both 100 and at 10 nm, increasing the
number of consecutive guide-strand amide linkages from three to
six resulted in progressively weaker silencing (Figure 2). Due to
this trend and the limited amount of G7 synthesized, G7 was not
included in these experiments. The results in Figure 2 showed
that siRNA having five total amide linkages (four in guide strand
G4 and one in passenger strand P1) had high silencing activity
(~80%) at 10 nM siRNA concentration; siRNAs with higher
number of amide modifications retained useful, albeit lower
silencing activity at 100 nM.
Similar results were observed when the amide-modified guide
strands were paired with passenger strand P2 having two amide
linkages at its 5´-end (Figure 3). While the activity of G3-P2 was
similar to that of the positive control G0-P2 at both 100 and at 10
nM, increasing the number of consecutive guide strand amide
linkages lead to progressively weaker silencing. Similar to results
obtained with P1-containing siRNAs in Figure 2, the results in
Figure 3 showed that G3-P2 having five total amide linkages
(three in guide strand G3 and two in passenger strand P2) had
high silencing activity (~80%) at 10 nM siRNA concentration,
while G4-P2 and G5-P2 retained useful, albeit lower silencing
activity at 100 nM.
duplexes (Table S2). A single amide modification caused a drop
in melting temperature from ~65 °C for G0-P0 to ~60 °C for G0-
P1. However, further increasing the number of amide linkages in
either guide or passenger strand did not significantly change the
thermal stability of siRNA duplexes, with melting temperatures
ranging from 60.1 to 61.6 °C. We concluded that decreases in
RNAi activity observed when increasing the number of amide
modifications did not correlate with changes in thermal stability of
the modified siRNA duplexes.
To test if the decreased negative charge due to multiple amide
modifications enabled passive uptake of siRNAs, we compared
the silencing activities of G7-P1 (8-amides), G7-P2 (9-amides),
and the unmodified G0-P0 (no amides) in the presence and in the
absence of transfection reagent (RNAiMAX) at 100 nM siRNA
duplex concentrations (Figure S15). As expected from the results
in Figures 2 and 3, in the presence of RNAiMAX the strong RNAi
activity of G0-P0 decreased with the increasing number of amide
linkages in G7-P1 and G7-P2. Nevertheless, G7-P2 achieved
~50% silencing of PPIB at 100 nM. There was no detectable
knockdown for any of the duplexes in the absence of RNAiMAX,
suggesting that elimination of up to nine phosphates (~22% of all
siRNA phosphates) was not sufficient to enable passive uptake of
amide-modified siRNAs.
Nuclease stability of amide-modified RNAs. Previous studies
by Iwase et al.[16] showed that two consecutive amide linkages at
the 3´-ends of both guide and passenger strands enhanced the
resistance of siRNAs against degradation by nuclease S1 and
50% mouse plasma. We observed similar enhancement of
stability of the 3´-amide-modified guide strands against 3´-
specific snake venom phosphodiesterase (SVPD) and the 5´-
amide-modified passenger strands against 5´-specific bovine
spleen phosphodiesterase (BSPD). For example, >90% of G3-G5
remained intact (Figure S20) under conditions where more than
50% of the unmodified G0 was digested by SVPD. Similarly, P1
and P2 were digested more slowly by BSPD than P0 or G3 that
lacked the 5´-amide modifications (Figure S21). Overall, these
results were consistent with Iwase et al.[16] and confirmed the
expected trend that amide-linkages increase the nuclease
stability of RNA.
Figure 3. Concentration-dependent silencing activity of amide-modified guide
strands G3, G4, G5, and G6 (for sequences, see Table 1) paired with passenger
strand P2 having two consecutive amide linkages at its 5´-end. Activity is
presented as a PPIB-to-GAPD mRNA ratio in HeLa (CCL2) cells, normalized to
a non-target control (NTC). G0-P2 is the positive control. The data are averages
of biological duplicate experiments, each performed as a technical triplicate.
Conclusions
In this study, we developed methods that allowed us for the first
time to introduce up to seven consecutive amide linkages
between uridine and adenosine nucleosides in RNA. Limitations
currently remain due to challenges associated with the synthesis
of cytosine and guanosine nucleoside amino acids, as well as
modest amide-coupling efficiencies. Synthesizing cytosine and
guanosine monomers and optimizing amide couplings remain
future tasks whose accomplishment will expand the sequences
that can be linked with amides and allow for the preparation of
longer stretches of amide-linked RNA. Nevertheless, the current
methodology allowed us to explore the limits of amide
modification in a model siRNA duplex. We found that up to five
amide linkages were tolerated without significant loss of RNAi
activity. An siRNA having four consecutive amide linkages in the
We have previously reported that isolated amide linkages did not
significantly change the structure and thermal stability of RNA
duplexes[13] and caused relatively small decreases in RNAi
activity when incorporated in an siRNA guide strand.[14a] Three
consecutive amide linkages caused relatively small changes in
the structure of an A-type duplex; however, the melting
temperature of the duplex was significantly lowered.[15] To test if
the observed decrease in RNAi activity in Figures 2 and 3
correlated with systematic changes in thermal stability, we
recorded the melting temperatures of the corresponding siRNA
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guide strand maintained high silencing activity (~80% of target
silencing) at 10 nM when paired with a passenger strand having
one amide modification between its first and second nucleosides.
A similar result was observed for an siRNA having three and two
consecutive amide linkages in the guide and passenger strands,
respectively. Further increases in the number of amide
modifications in either the guide or passenger strand decreased
RNAi activity; however, siRNA duplexes with up to nine amide
linkages retained useful RNAi activity at higher (100 nM) siRNA
concentrations. It should be noted that the siRNA duplex studied
herein showed high sensitivity to amide modifications in one of
our previous studies.[14a] It is conceivable that other siRNA
sequences may be more tolerant to multiple amide substitutions.
While an siRNA duplex having nine amide linkages retained some
silencing activity in the presence of lipid transfecting agent, we did
not detect any activity in the absence of transfection agent,
indicating that the partial reduction of the negative charge did not
enable passive uptake in HeLa cells. Taken together our results
suggest that multiple consecutive amide linkages may be used to
optimize biological properties of siRNAs. However, additional
chemical modifications, such as conjugation with cell-penetrating
peptides, are needed to enable cellular uptake of siRNAs in the
absence of transfection agents.
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Detailed methods are described in the Supporting Information.
Acknowledgements
This work was supported by the National Institutes of Health (R01
GM071461 and R35 GM130207 to E.R.). The Regional NMR
Facility (600 MHz instrument) at Binghamton University is
supported by NSF (CHE-0922815).
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Keywords: RNA interference • chemical modifications •
backbone modified RNA • internucleoside amide linkage • non-
ionic RNA analogues
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10.1002/chem.201903754
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An siRNA having four consecutive
phosphates of its guide strand
replaced by amide linkages showed
high silencing activity when the
passenger strand had an amide
linkage between its first and second
nucleosides. Further increase in
amide modification decreased the
RNAi activity; however, siRNAs
having up to nine amide linkages
retained some silencing activity at
higher concentrations.
Venubabu Kotikam,* Julien A. Viel and
Eriks Rozners*
Page No. – Page No.
Synthesis and Biological Activity of
Short Interfering RNAs Having
Several Consecutive Amide
Internucleoside Linkages
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