Synthesis, biophysical studies and RNA interference activity of RNA having three consecutive amide linkages

Article abstract of DOI:10.1039/c3ob42532k

RNA sequences having up to three consecutive internal amide linkages were synthesized and studied using UV and NMR spectroscopy. The amide modifications did not interfere with normal base-pairing and A-type RNA conformation. Three consecutive amides were well tolerated in the passenger strand of siRNA and caused little change in RNAi activity.

Full text of DOI:10.1039/c3ob42532k

Organic &
Biomolecular Chemistry
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Synthesis, biophysical studies and RNA
interference activity of RNA having three
consecutive amide linkages†
Cite this: Org. Biomol. Chem., 2014,
12, 1207
Received 18th December 2013,
Accepted 6th January 2014
Paul Tanui,a Scott D. Kennedy,b Benjamin D. Lunstad,c Amanda Haas,c Devin Leakec
and Eriks Rozners*a
DOI: 10.1039/c3ob42532k
www.rsc.org/obc
RNA sequences having up to three consecutive internal amide
linkages were synthesized and studied using UV and NMR spectro-
scopy. The amide modifications did not interfere with normal
base-pairing and A-type RNA conformation. Three consecutive
amides were well tolerated in the passenger strand of siRNA and
caused little change in RNAi activity.
RNA interference (RNAi) has become a powerful and widely
used technology for functional genomics and has intriguing
potential for becoming a new therapeutic strategy.1,2 However,
for in vivo applications in RNAi technologies the oligoribo-
nucleotides need chemical modifications for optimization of
properties such as enzymatic stability, targeted delivery, bio-
distribution and pharmacokinetics, and cellular uptake.1,2
While numerous good solutions for improving enzymatic
stability have emerged, optimization of the latter properties
remains challenging. Phosphorothioate (PS, Fig. 1), where the Fig. 1 Structure and retrosynthetic analysis of amide-modified RNA.
non-bridging oxygen is replaced with sulfur, is one of a few
modifications that enhances binding of antisense oligonucleo-
Replacement of phosphodiesters with non-ionic amide lin-
kages (such as AM1, Fig. 1) has been explored as a means to
increase enzymatic stability of antisense oligodeoxynucleo-
tides.7 DNA having isolated AM1 modifications hybridized to
complementary RNA with affinity similar to the unmodified
DNA.8,9 NMR10 and molecular modeling9,11 studies suggested
that AM1 linkages were well accommodated in an A-form
DNA–RNA heteroduplex and favored an A-form conformation
of the modified DNA. Von Matt et al. showed that DNA having
several consecutive AM1 linkages had somewhat lower affinity
(Δtm ca. −0.5 °C per modification) for the complementary RNA
than the unmodified control.12
tides to serum albumin, which improves pharmacokinetics
and circulation time of the modified oligonucleotides.3 PS lin-
kages are well tolerated in short interfering RNAs (siRNAs),
especially at the ends of duplexes, and lead to more persistent
gene silencing.4–6 While a fully modified siRNA (40 PS lin-
kages) was not toxic in one study,4 others have observed toxi-
city of siRNAs with 12 and 20 PS modifications.5,6 Although
these studies suggest that modification of the phosphodiester
backbone has strong potential for optimization of siRNAs,
such an approach has been little explored beyond the PS
modification.
The relatively simple synthesis and favorable RNA binding
of amide-modified DNA suggested that AM1 might also hold
promise for optimization of siRNA properties. Robins, Peter-
son, and co-workers pioneered the synthesis of AM1-linked
RNA dimers13 and pentamers;14 however, the biophysical pro-
perties of these analogues were not reported. Later, we demon-
strated that isolated AM1 linkages had little effect on the
structure, thermal stability and hydration of RNA double
aDepartment of Chemistry, Binghamton University, The State University of New York,
Binghamton, New York 13902, USA. E-mail: erozners@binghamton.edu
bDepartment of Biochemistry and Biophysics, University of Rochester School of
Medicine and Dentistry, Rochester, New York 14642, USA
cGlobal Research and Development in Molecular Biology, Thermo Fisher Scientific
Bioscience Division, Lafayette, Colorado 80026, USA
†Electronic supplementary information (ESI) available: Experimental procedures
and results, and copies of 1H and 13C NMR data. See DOI: 10.1039/c3ob42532k
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helices.15,16 Iwase and co-workers17 found that two consecutive The 2′-O-Ac protected 2a was prepared from D-xylose following
amides at the 3′ overhangs (prepared using Robin’s methods) our recently developed procedure.18 The 2′-O-TBS protected 2b
increased the enzymatic stability of siRNAs, but did not impair was prepared from the known 6.18 Protecting group exchange
their RNAi activity. Taken together, our preliminary results and oxidation gave the azido acid 7b,19 which was converted to
and Iwase’s encouraging studies prompted us to explore the 2b using the same procedures as for 2a.18 Modified uridines
structure and biological properties of RNA harboring con- 3a (R = Ac) and 3b (R = TBS) required for switching from
secutive amides. Herein, we report that three consecutive AM1 amide to phosphodiester synthesis were made as previously
linkages somewhat decreased the thermal stability but main- reported by us.15,16
tained the A-form conformation of an RNA helix. When placed
Our initial experiments focused on preparing the amide-
close to the 5′-end, the amide modifications were well tolerated modified RNAs (Fig. 1) using monomers 1, 2a and 3a. A brief
in the passenger strand of siRNA. To our knowledge, this is study of amide coupling conditions revealed that using 10
the first time that the effect of three consecutive non-ionic lin- equivalents of 2a (0.125 M in DMF) activated by HATU in the
kages on the structure and biological activity of RNA has been presence of diisopropylethylamine gave ∼90% yields after
characterized. The results suggest that amides are excellent 8 hours. However, attempts to introduce amides at either 3′- or
mimics of the phosphate backbone in RNA, are well tolerated 5′-ends of siRNAs gave only phosphodiester-linked RNA frag-
in biological systems, and may have the potential to optimize ments that were missing the amide-linked portions and termi-
properties of siRNAs for in vivo applications.
nated at the transition monomers 1 and 3. In other words, the
Synthesis of RNA having consecutive internal amide lin- amide linkages appeared to have degraded during the post-
kages required three types of modified nucleosides, uridine synthetic manipulation. In our earlier studies we noted
derivatives in our case (Fig. 1): (1) 5′-aminouridine-3′-phos- decreased stability of amides in RNA (presumably due to the
phoramidite 1 to switch from standard RNA to amide-linked 1,5 relationship of carbonyl and 2′-OH) and optimized the
RNA synthesis; (2) 5′-amino-3′-carboxymethyluridine
extend the amide backbone; and (3) 3′-carboxymethyluridine 3 methanol (2 days at rt) did not cause amide cleavage.19 The
to switch back to standard RNA synthesis. apparent degradation of consecutive amides in RNA was sur-
2 to deprotection conditions; we found that using ammonia in
The approach for RNA1, for example, was to start with prising because we had successfully synthesized RNAs having
standard RNA synthesis using commercially available isolated amides using the 2′-O-Ac protected 3a.16
a
phosphoramidites to assemble a stretch of phosphodiester
Although the precise mechanism of the unexpected amide
backbone, 5′-CpGpC then, after introducing monomer 1, degradation remained unclear, using the 2′-O-TBS instead of
build a stretch of amide backbone using monomers 2, 2′-O-Ac enabled synthesis of intact amide-modified RNAs. The
5′-UaUaUpCpGpC, and finally, after adding 3, complete the synthesis started as a standard automated phosphoramidite
5′-GpCpGpUaUaUaUpCpGpCp sequence using standard phos- protocol (1 μmol on Expedite 8909) concluding with monomer
phoramidite chemistry.
1. The synthesis column was then removed from the synthesi-
Compound 1 was prepared using standard nucleoside zer and the amide portion was synthesized manually using 2b
chemistry (Scheme 1). For construction of consecutive amide as described in ESI.† After manual addition of 3b, automated
linkages, we explored two nucleoside amino acids, 2a and 2b. RNA synthesis finished preparation of RNA1–RNA4 (Fig. 1).
The overall yields of amide-modified RNA were good for RNA1
(2 to 5%) but relatively poor for the longer RNA2–RNA4 (0.2 to
1.2%). MS analysis identified phosphodiester-linked RNA frag-
ments terminated with acetylated monomer 1 (capped RNAs
missing the amide-linked portions) as the major impurities
(for details, see ESI†). These results suggested that either
inefficient amide couplings or residual degradation of the
amide backbone might be responsible for decreased overall
yields. Nevertheless, the modest efficiency (the synthesis was
not thoroughly optimized) was adequate for the NMR and
RNAi activity studies.
UV thermal melting studies showed that three consecutive
amide linkages in RNA1 lowered the thermal stability of the
decamer duplex by 3.5 °C per modification (Table 1). Neverthe-
less, CD (Fig. S8†) and NMR spectroscopy studies showed that
Scheme 1 Synthesis of protected uridine 5’-amino 3’-C-carboxylic RNA1 formed a typical A-form duplex despite the consecutive
amide modifications. As in our previous study,16 osmotic
acids. Steps: (a) Bu2SnCl2, diisopropylethylamine, dichloroethane, rt, 1 h,
then TOM-Cl, 80 °C, 0.5 h, 36%; (b) H2S gas, pyridine–water, 4 : 1, 1 h,
then stirred overnight, 88%; (c) methoxytrityl chloride, pyridine, rt, over-
night, 77%; (d) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, di-
stress20 (Table 1, last three columns) showed that amide modi-
fications caused little change in RNA hydration.
1D and 2D NMR spectra of the imino protons indicated
that all expected Watson–Crick pairs were formed, including
isopropylethylamine, CH2Cl2, rt, 14 h, 61%; (e) ref. 18; (f) ref. 19; (g) ref.
18.
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Table 1 UV melting and osmotic stress of duplexes formed by RNA1 and unmodified control
−ΔHb
−ΔHc
−ΔHd
−ΔG310
Ethylene
glycol ΔnW
Glycerol Acetamide
d
Sequencea
tm (°C)
(kcal mol−1
)
(kcal mol−1
)
(kcal mol−1
)
ΔSd eu
209
(kcal mol−1
)
ΔnW
ΔnW
e
e
e
5′-GCGUaUaUaUCGC
3′-CGCApApApAGCG
48.6 0.2 74.0 3.0
86.0
7
76.1 2.0
96.6 4.4
6
11.5 0.3
46
52
3
4
59
5
7
60
5
4
5′-GCGUpUpUpUCGC 59.1 0.3 87.2 2.7
3′-CGCApApApAGCG
116 19
263 13 15.1 0.3
63
66
a RNA strands, 2 μM each in 10 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA, and 300 mM NaCl. Results standard deviations. b From
dα/d(1/Tm) vs. Tm. c From concentration dependency of Tm. d From van’t Hoff analysis. e ΔnW = number of waters released upon melting.
Results error estimates.
Fig. 2 NMR data and model of duplex formed by RNA1 and a comp-
lementary RNA strand: (A) 1D imino and 2D NOE spectrum of RNA1 indi-
cates formation of four AU pairs. (B) Model (green) overlaid with PDB
1H1K (grey, all AU pairs), the dominant orientation of the amide linkages
is indicated.
the four AU pairs flanking amide modifications (Fig. 2A). Con-
sistent with the UV melting data, weaker NOE cross-peaks for
the central AU pairs compared to the flanking AU pairs,
especially at room temperature, suggested reduced stability
towards the center of the duplex (Fig. S9†). All riboses were in
C3′-endo conformation, typical of A-form RNA (Fig. S10†).
Amide protons show very weak or missing NOEs to H2′ and
H3′ of the residue 5′ of the amide and medium NOEs to H4′ of
the 3′-residue (Fig. S11†). The NOE patterns were similar to
those observed between the amide protons and sugar protons
in a duplex with isolated amide linkages studied previously
Fig. 3 RNAi activity of siRNAs formed with amide-modified passenger
strand in HeLa cells: (A) RNA2 and (B) RNA3 and RNA4. Control and UT
by us.16 Comparison of a representative model resulting
from simulated-annealing with a relatively small number of
restraints with the crystal structure of an all AU-pairs RNA
duplex revealed that the three amide modifications caused
notable, but relatively small deviations from the A-form confor-
mation. The dominant conformation of the amide linkages
oriented the CvO bond towards the major groove and NH
towards the minor groove (Fig. 2B) and was consistent with
previous molecular dynamics11 and our recent NMR16 studies.
Finally, the effect of consecutive amide modifications on
RNAi activity was tested using RNA2–RNA4, which is a passen-
ger strand of siRNA targeting the Cyclophilin B (PPIB) mRNA.
Introduction of three consecutive amides near the 5′-end
represents unmodified siRNA and lipid-only treated cells, respectively.
caused very little change in the silencing activity of RNA2
(Fig. 3A). This result was both encouraging and perhaps un-
expected because Gong and Desaulniers have recently reported
that an amide linkage derived from insertion of a PNA
monomer was not well tolerated near the 5′-end of siRNA’s
passenger strand.21 The somewhat higher activity of RNA4
compared to RNA3 (Fig. 3B, siRNAs having two consecutive
amide linkages) suggested that an amide modification at the
third position may help loading of the guide strand, but
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further testing is needed to confirm this. Overall, the data indi-
cate that consecutive amides are well tolerated in biological
systems.
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We thank NIH (R01 GM071461) for financial support of this
research. The Regional NMR Facility (600 MHz instrument) at
Binghamton University is supported by NSF (CHE-0922815).
We thank Daniel Mutisya for synthesis of 3b.
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