Synthesis and Characterization of Thiophosphoramidate Morpholino Oligonucleotides and Chimeras

Article abstract of DOI:10.1021/jacs.0c04335

This Article outlines the optimized chemical synthesis and preliminary biochemical characterization of a new oligonucleotide analogue called thiophosphoramidate morpholinos (TMOs). Their rational design hinges upon integrating two well-studied pharmacopho

Full text of DOI:10.1021/jacs.0c04335

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Article
Synthesis and Characterization of Thiophosphoramidate
Morpholino Oligonucleotides and Chimeras
Heera K Langner, Katarzyna Jastrzebska, and Marvin H. Caruthers
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c04335 • Publication Date (Web): 31 Aug 2020
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Journal of the American Chemical Society
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Synthesis and Characterization of Thiophosphoramidate Morpholino
Oligonucleotides and Chimeras.
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Heera K. Langner*, Katarzyna Jastrzebska†, Marvin H. Caruthers*
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309, United
States
ABSTRACT: This manuscript outlines the optimized chemical synthesis and preliminary biochemical characterization of a
new oligonucleotide analogue called thiophosphoramidate morpholinos (TMOs). Their rational design hinges upon
integrating two well-studied pharmacophores, namely phosphorothioates (pS) and morpholinos, to create morpholino-pS
hybrid oligonucleotides. Our simple synthesis strategy enables the easy incorporation of morpholino-pS moieties and
therapeutically relevant sugar modifications in tandem to create novel oligonucleotide (ON) analogs that are hitherto
unexplored in the oligotherapeutics arena. Exclusively TMO modified ONs demonstrate high stability towards 3’-exonuclease.
Hybridization studies show that TMO chimeras consisting of alternating TMO and DNA-pS subunits exhibit higher binding
o
affinity towards complementary RNA relative to the canonical DNA/RNA duplex (~10 C). Oligonucleotides that consist
entirely of TMO linkages also show higher RNA binding affinity, but do not recruit RNase H1. Chimeric TMO analogues
demonstrate high gene silencing efficacy, comparable to that of a chimeric 2’-OMe-pS/pO control, during in vitro bioassay
screens designed to evaluate their potential as microRNA inhibitors of hsa-miR-15b-5p in HeLa cells.
Introduction
has gained substantial interest. Two steric blocker PMO
drugs which can access a “difficult to drug” target, namely
the mRNA of the human dystrophin gene, have been
conditionally approved for the treatment of Duchenne
Muscular Dystrophy and two more are under Phase III
clinical trials.8-10 Several oligonucleotide drugs containing
the phosphorothioate linkage have also been FDA approved
(Vitravene, Kynamro, Spinraza, Tegsedi™, Givlaari™).
During their course of development, the clinically relevant
pharmacokinetics, biodistribution, metabolism and toxicity
profiles of both pS and PMO based ONs have undergone
intensive research validation through many years of
elaborate testing and clinical trials.11 These studies have
revealed that PMOs exhibit fast plasma clearance and
minimal drug related toxicity at clinically relevant doses;
however, they also show poor cellular uptake and
pharmacokinetics.12 In contrast, ONs containing pS
internucleotide linkages interact with a wide variety of
proteins (e.g., plasma proteins) resulting in off-target
effects and toxicity.13 Although they possess superior
enzymatic stability, their “stickiness” leads to reduced renal
clearance, longer circulation half-lives and good tissue
distribution.14
In this age of personalized medicine, oligonucleotide
therapies are emerging as a reliable platform for drug
development.1 Due to their size (between 6-10 kDa) and
charge, oligonucleotides (ONs) possess very different
pharmacodynamic and pharmacokinetic properties relative
to conventional small molecule drugs. While small
molecules drugs directly interact with diseased proteins,
ON drugs alter the mRNAs that produce these proteins and
thereby prevent pathogenesis at a much earlier stage.2
Synthetic ON drugs can interact specifically with their
mRNA target through Watson-Crick base pairing and either
force RNA blockade (e.g., antisense-mediated exon-
skipping, microRNA inhibition) or irreparably degrade their
target (e.g., RNase H1 or Ago2 mediated cleavage) which
stops the transfer of genetic information from the mutated
gene to the defective protein.3-4 Their unique modes of
action enables them to target the “undruggable” space,
which includes several rare genetic disorders for which no
therapeutic modalities currently exist.5
Depending upon structural design and how they alter
protein expression, ON drugs are classified into antisense,
siRNA, antagomirs, splice-switching oligonucleotides,
aptamers and so on.6 Over the past four decades, numerous
chemical modifications have been developed to modulate
various biochemical properties such as binding affinity,
nuclease resistance, in vitro and in vivo delivery and to
minimize immune stimulation and associated toxicities.7
Among these analogues, one distinct class of synthetic ONs
called phosphorodiamidate morpholinos (PMOs, Figure 1a)
Despite their individual successes, the chemical synthesis of
oligonucleotide hybrids that incorporate both morpholino
and DNA-pS subunits has been hampered by the inherent
drawbacks associated with utilizing the P(V) based
synthetic strategy that is currently being used to generate
PMOs.15 Additionally, second and third generation sugar
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phosphoramidates with P=S might reasonably improve
1
2
3
4
5
6
7
8
their hydrolytic stability under acidic conditions and also
increase their nuclease resistance towards intracellular
enzymes. This rationale has led us to focus entirely on ONs
containing thiophosphoramidate morpholinos (TMO,
Figure 1c) and TMO chimeras containing DNA-pS linkages
(Figure 1d). In this article, we evaluate the stability of TMO
analogues towards aqueous acid, their hybridization
affinity towards complementary DNA and RNA as well as
their ability to recruit RNase H1. We further assess their
biological potency as microRNA-15b inhibitors using a dual-
luciferase reporter assay.
O
B
B
B
O
O
O
O
O
N
O
N
P N
O
O
P X
O
O
P SH
O
O
B
B
B
O
O
O
N
N
N
O
P X
O
O
P SH
O
P N
O
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O
(1d)
X = O (1b) or S (1c)
(1a)
Results and Discussion
Figure 1 Chemical representation of phosphorodiamidate
morpholino (PMO) (1a), phosphoramidate morpholino (1b),
thiophosphoramidate morpholino (TMO) (1c) and TMO-DNA-
pS chimeras (1d).
Chemical Synthesis
Morpholino nucleosides of N6-benzoyl adenosine (mABz),
N4-benzoyl cytidine (mCBz), N2-isobutyryl guanosine
(mGiBu) and thymidine (mT) (compounds 3a-d) were
synthesized according to published literature protocols
(Scheme 1).21-22,26 Briefly, 7.42 mmol of 5’-dimethoxytrityl
(DMT) protected ribonucleoside (5.0 g (2a), 4.87 g (2b),
4.82 g (2c) or 4.16 g (2d)), was dissolved in 500 mL of
anhydrous methanol followed by the sequential addition of
1.2 equiv. of sodium periodate (1.9 g, 8.9 mmol) and 1.2
equiv. of ammoniumbiborate tetrahydrate (2.35 g, 8.9
modifications such as 2’-OMe, 2’-F, 2’-O-MOE, and LNA16 are
only available as phosphoramidite synthons. Although
several PMO hybrids have been synthesized using
conventional P(V) chemistry,17
a robust and flexible
synthetic route that would generate chimeras containing
morpholino nucleotides in conjunction with therapeutically
relevant modifications has yet not been reported. Such
chimeric analogues could prove beneficial for developing
new therapeutic drugs with biological attributes that are
unlike those of their parent pharmacophores.
o
mmol). The mixture was stirred at 25 C for 6 h or until no
unreacted ribonucleoside could be detected by TLC. The
reaction mixture was filtered through a pad of Celite, and
to the filtrate was added
2
equiv. of sodium
Towards this goal, previous work has focused on the
synthesis of morpholino-DNA chimeras containing various
chemical modifications including boranophosphoramidate,
phosphoramidate and alkyl phosphoramidate linkages.18
cyanoborohydride (0.93 g, 14.8 mmol) and 2 equiv. of
glacial acetic acid (0.85 mL, 14.8 mmol). The reaction
mixture was stirred for 16 h, filtered, and the solvent was
removed using a rotary evaporator. The crude reaction
mixture was purified by chromatography on a silica gel
column pre-equilibrated with ethylacetate (EtOAc)
containing 3% triethylamine. The column was first eluted
with 100% EtOAc followed by EtOAc:Methanol (70:30) to
yield the 6’-DMT27 protected morpholino nucleosides (3a-
d): mABz (2.75 g, 56%), mGiBu (2.21 g, 46%), mCBz (2.40 g,
51%) and mT (2.33 g, 58%).
Other
literature
examples
include
and
morpholino
triazole-linked
methylphosphonamidates19
morpholino analogs.20 Zhang et al. reported the synthesis
and characterization of phosphoramidate morpholino-DNA
chimeras (10-12mers) containing up to three morpholino
thymidine (mT) phosphoramidate linkages (Figure 1b) and
morpholino-RNA chimeras (21 mers) having one or two
morpholino uridine (mU) phosphoramidate linkages.21-22
They reported that the introduction of one or more
morpholinophosphoramidate linkages resulted in reduced
hybridization affinity relative to an unmodified DNA/RNA
heteroduplex. Notably, when siRNA duplexes containing
one or two terminal morpholinophosphoramidate linkages
(3’- and 5’- ends) were treated with serum, their stabilities
Zhang et al. have reported the chemical synthesis of
morpholino phosphorodiamidites of mU and mT.21-22
Following slight modifications to these protocols,
phosphorodiamidites of mABz, mGiBu, mCBz and mT (4a-d,
Scheme 1) were synthesized.26 Briefly, one equivalent of
each morpholino nucleoside (2.6−3.1 g, 4.8 mmol) and 1.2
equivalents of 2-cyanoethyl N,N,N’,N’-tetraisopropyl
phosphorodiamidite (1.83 mL) was reacted with 0.5
equivalents of 0.25M 5-(Ethylthio)-1H-tetrazole (ETT, 9.6
mL) in dichloromethane for 30 minutes. After evaporating
to dryness, the crude product was quickly purified by
column chromatography. The pure phosphorodiamidites
were isolated in moderate yields (4a: 2.75 g, 67%, 4b: 2.37
g, 59%, 4c: 2.63 g, 66%, and 4d: 2.93 g, 82%). The detailed
synthetic protocol and characterization data (1H, 31P, 13C,
ESI-MS) for all morpholino phosphorodiamidites are
provided in the Supporting Information (SI, Section 2 and SI
p.S45-S48).
improved
4
to 7-fold relative to unmodified
oligonucleotides. Improved serum stability has also been
observed with other phosphoramidate analogues.23-24 Of
particular interest was the observation that siRNA duplexes
containing morpholino phosphoramidate subunits were
active at nanomolar concentrations in a dual luciferase
reporter assay.
From a chemical perspective, the solid-phase synthesis of
morpholinophosphoramidate ONs resulted in poor overall
yields. Their crude reaction mixtures could not be purified
using the conventional DMT-On/Off procedure due to their
inherent instability to aqueous acid.25 We hypothesized that
replacing a non-bridging P=O linkages in morpholino
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O
B
DMTO
O
B
DMTO
B
DMTO
B
DMTO
1
O
O
(iii)
(i)
(ii)
N
P
2
3
4
5
6
7
8
9
N
N
H
N
O
O
O
OH
2a-d
OH
4a-d
Yields: 59-82%
3a-d
Yields: 46-58%
B = N6-Benzoyladenosine (2a)
N2-isoButyrylguanosine (2b)
N4-Benzoylcytidine (2c)
Thymidine (2d)
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Scheme 1 Chemical synthesis of phosphorodiamidite synthons 4a-d. Reagents and conditions: (i) NaIO4 (1.2 equiv.),
anhydrous methanol; (NH4)2B2O7 (1.2 equiv.), 6 h (ii) NaCNBH3 (2.0 equiv);CH3COOH (2.0 equiv.), 16h (iii)
(OCH2CH2CN)P[N(iPr)2]2 (1.2 equiv.), 5-Ethylthio-1H-Tetrazole (0.5 equiv.),CH2Cl2, 30 min.
Optimization of TMO Synthesis
To summarize, the lack of highly selective activation of
the N,N’-diisopropylamino group versus the 6’-DMT-
morpholinonucleoside can lead to an exponential loss of
yields during the synthesis of mixmer TMOs for biological
Unlike conventional nucleoside phosphoramidites of the
general formula (R1O)(R2O)P[(N,N’-(iPr)2] (Figure 2, A),
where only one P(III)-N linkage is activated during the
coupling step,28 phosphorodiamidite morpholino synthons
applications (≥ 25 mers). Therefore, an important step
(4a-d) contain two P(III)-N linkages (Figure 2, B) both of
during coupling optimization was to determine which
which can get activated to different degrees depending
activator generated the highest yield of final product while
upon the reaction conditions. Barone et al. demonstrated
minimizing the amount of truncated failures and associated
that when a phosphorodiamidite comprising two amino
side products.
substituents, i.e., P-Methoxy-(N,N’-diisopropylamino,N-
In
order
to
address
this
problem,
several
morpholino) phosphorodiamidite, was reacted with 1H-
tetrazole, activation of the N,N’-diisopropylamino group
was the major pathway, leading to the formation of
morpholino substituted P-Methoxy phosphoramidite as the
major product (95%), with only 5% of morpholino
activated product.29a Since protonation of the amine leaving
group and weakening of the P-N bond is the first step during
dodecathymidylate TMOs were synthesized under identical
conditions except that different activators were used during
the coupling step. Since the poor yields could be mainly due
to competing activation of the 6’-DMT-morpholino subunit,
all activators were tested at 50% of their manufacturer
recommended concentrations. We anticipated that an
increased coupling time of 600 seconds might compensate
for any loss of reactivity owing to the lower activator
concentrations. Post synthesis, the ratio of full-length
product versus failures in the total reaction mixture was
calculated by integrating their UV profiles. The ratio of the
expected product (TMO1, Table 1) in each case is shown in
SI Figure S1.
phosphoramidite
activation,
the
more
basic
diisopropylamino group (pKa = 11.07, water) has a greater
propensity for protonation compared to the morpholino
ring nitrogen (pKa = 8.49, water), making it a superior
leaving group. Thus, previous studies comparing 1H-
tetrazole-mediated
activation
of
5’-DMT-2’-
deoxythymidine phosphoramidites containing either N-
morpholino or N,N’-diisopropylamino as the amine leaving
group showed 95% activation of the diisopropylamino
group within 1 minute, whereas only 5 % activation of the
morpholino group was observed under the same
conditions,29b suggesting that the morpholino ring nitrogen
might be less susceptible to activation when compared to
the N,N’-diisopropylamino group.
During TMO synthesis, the coupling step must proceed by
protonation of the phosphorodiamidite followed by
displacement of one of the amine leaving groups resulting
in
a reactive intermediate which further undergoes
nucleophilic attack.30 We first investigated the effect of high
acidity (faster protonation rate) on amine selectivity. The
lowest yields (61.7%) were obtained for the highly acidic 5-
[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator
42, pKa = 3.4) (SI Figure S1a). This could be the result of
competing morpholino activation leading to premature self-
capping of the growing TMO chain as a 5’-N,N’-diisopropyl
phosphoramidate. Additionally, 6’-DMT detritylation,
Previous studies have shown that the specificity of
activation of the morpholino ring nitrogen versus the N,N’-
diisopropylamino group was dependent on the activator.
When 0.5 equivalents of ammonium tetrazolide salt was
used to activate P-Methoxy-(N,N’-diisopropylamino,N-
morpholino) phosphorodiamidite, 10% of morpholino
activated product was observed29a as opposed to only 5%
morpholino activated product while using 1H tetrazole.29b
In line with this observation, our preliminary attempts to
synthesize TMOs using 0.45 M 1H-tetrazole or 0.25 M ETT
resulted in poor overall yields and the concomitant
formation of large amounts of side products.
stepwise
double
activation
of
the
incoming
phosphorodiamidite or degradation of the TMO backbone
due to the high acidity of Activator 42 may occur during
the long coupling step (600s wait).
Low yields (65.6%) and large amounts of failure products
were also observed when 0.12 M 1H-tetrazole (TET, pKa =
4.9) was used. The presence of a significant amount of very
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1
2
3
4
5
6
7
8
DMTO
O
HO
H
N
H
N
B
DMTO
O
(i)
O
O
O
N
B
O
HO
O
N
N
Cl2HC
O
R
H
Cl2HC
O
O
H
DMTO
B
DMTO
O
O
P
OR1
O
S
USIII
B
(ii)/B,(ox),(i)
(i)
B
O
S
HO
O
O
H
N
O
P
R
O
O
P
O
R
O
HO
R
O
HO
B
DMTO
N
O
B
O
O
B
Cl2HC
O
S2
H
DMTO
or
O
B
O
O
O
S1
(i)
or
N
P
N
P
O
N
P
O
P
HO
S
Next cycle
N
OR1
HO
S
OR1
N
O
B
O
B
O
9
A
B
N
H
10
11
12
13
14
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19
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22
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R
OH
G
H
I: (v)
J: (vi)
S1
S2
C
D
(ii)/B
I
J
O
B
DMTO
DMTO
R1O
B
N
P
O
S
O
B
OR1
DMTO
O
N
B
O
R
O
N
P
P
O
B
DMTO
B
R1O
O
O
E
F
G
H
B
O
or
O
O
P
OR1
O
(iii), (iv)
(i),(ii/A), (iii),(iv)
N
P
S
O
H
N
O
R1O
O
R
O
B
R
O
O
R1O
P
S
O
O
N
P
O
O
O
B
N
Cl2HC
O
H
or
O
OR1
O
O
B
DMTO
CHCl2
D
C
N
P
O
N
P
R1O
S
H
O
O
O
B
N
DMTO
S
OR1
B
H
N
O
O
N
B
O
N
P
O
O
R1O
S
R
O
B
O
P
OR1
O
or
O
H
G
O
O
H
N
R
O
O
O
R = OMe, H
= CH
R1
2CH2CN
O
N
Cl2HC
O
H
F
E
Figure 2 Solid-phase Synthesis of TMO and TMO-DNA-pS Chimeras; Reagents: (i) Detritylation: 3% trichloroacetic acid/CH2Cl2 (ii)
Condensation: 0.12M 5-Ethylthio-1H-tetrazole/CH3CN, 600s wait (iii) Sulfurization: 0.05 M DDTT/pyridine/CH3CN (iv) Capping:
Ac2O/pyridine/THF; 1-Methylimidazole/CH3CN (v) Deprotection: 28% aqueous ammonia, 55 C, 16 h or (vi) Deprotection: 2M
ammonia in methanol, 60 min, followed by 28 % aqueous ammonia, 55 oC, 16 h. Amidites A, B are color-coded to simplify the cycle.
o
and higher percentage of morpholino activation, using 1.0
equiv. or more of ETT resulted in step-wise activation of
both amino substituents and produced symmetrical
dithymidine phosphotriester in significant amounts.
short failure products (Rt = 1.5−7 min; SI Figure S1c)
suggests the lower stepwise coupling efficiency of TET.
Since 5-Ethylthio-1H-Tetrazole is more acidic than 1H-
tetrazole (ETT, pKa = 4.3), the following conditions were
tested: 0.12 M ETT buffered with 0.01M DMAP31 (SI Figure
S1b) and 0.12 M ETT (SI Figure S1e). Surprisingly, the
highest yield of full-length product was obtained for
unbuffered, 0.12 M ETT (85.9%). The less acidic, more
nucleophilic 4,5-dicyanoimidazole (DCI, pKa = 5.2) also
produced TMO1 in relatively high yields (0.12 M DCI,
82.3%; SI Figure S1d). Attempts to increase the
nucleophilicity by using DMAP-buffered ETT produced the
expected product in lower yields compared to unbuffered
ETT (SI Figure S1b, 79.6%). These observations suggest that
apart from activator pKa, several other parameters such as
nucleophilicity, coupling time and activator concentration
play a major role in determining stepwise yields during
TMO synthesis. Based on the above results, 0.12 M ETT was
chosen as the activator of choice for solid-phase synthesis.
In summary, the solid-phase synthesis of TMOs and their
chimeras (Figure 2) reported here closely follows
conventional phosphorothioate DNA synthesis with only
two differences: (i) ETT concentration was reduced to 0.12
M instead of the manufacturer recommended 0.25 M (ii) a
600 s coupling time was used. For synthesizing TMO-DNA-
pS chimeras, a 30 second coupling time was used for
commercially
obtained
2’-deoxyribonucleotide
3’-
phosphoramidites. The detailed solid-phase synthesis
protocol is summarized in the SI, Section 3.
Optimization of DMT-On/Off Purification
Previous studies on the kinetic and mechanistic aspects
of hydrolysis of 3’,5’-thiophosphoramidates under a wide
pH range (pH 1-9) have shown that protonation of the 3’-
nitrogen generates a N(H)-P(V) linkage which is susceptible
to degradation at low pH (pH < 5). 32-33 Since TMO subunits
contain a P(V)-(N-morpholino) linkage, we evaluated the
hydrolytic stability of TMO1 (Table 1) under acidic pH.
TMO1 (Table 1) was treated with various concentrations of
aqueous acetic acid and the rate of degradation over time
To elucidate the role of activator concentration (0.25−1.5
equiv.) on product selectivity, the solution-phase reactivity
of 6’-DMT-morpholinothymidine phosphorodiamidite (4d,
Scheme1) with 3’-O-acetyl thymidine was monitored using
31P NMR as described elsewhere.25 While lower ETT
concentrations (0.25 equiv.) resulted in sluggish reactions
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Table 1. List of Oligonucleotides and Their Respective Yields**
1
2
3
4
5
6
7
8
9
ON#
Sequence Design*
Yields
Theoretical
Mass
Observed Massǂ
nmoles (µg)
1
6’-DMT-TTT TTT TTT TTt-3’
275.9 (990.6)
4053.6519
4948.8879
7061.0162
7074.7924
7071.8403
7390.8795
4942.6956
4941.7116
5670.8291
5066.8240
5050.8468
−
4053.659
4948.897
7061.029
7074.806
7071.854
7390.894
4942.704
4941.721
5670.839
5066.819
5050.843
−
2
6’-DMT-AGT TGC CCT GTG dTFl -3’
6’-T GTA AAC CAT GAT GTG CTG CTa-3’
6’-T GTA aac cat gat gtg ctG CTa-3’
5’-tGtA aAc CaT gAt GtG cTg Cta-3’
6’-T gTaA aCc AtG aTg TgC tGc Tat-3’
6’-Fls-TT TTT TTT TTT TTt-3’
138.0 (682.9)
46.8 (315.8)
3
4
117.0 (789.5)
88.9 (600.0)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
6
117.0 (789.5)
202.7 (1021.4)
181.7 (915.7)
164.1 (888.7)
622.1 (3134.6)
650.1 (3275.5)
807.7 (3823.7)
829.9 (3929.0)
70.4(433.7)
7
8
6’-Fls-TT TTT TTT TTT TTT-3’
9
6'-ATC CAA CTT ACC GTG ATc-3'
5’-Fls-tptptp tptptp tptptp tptptp tptT-3’
5’-Fls-tptptp tptptp tptptp tptptp tptpT-3’
5’-Fls-ttt ttt ttt ttt tt -3'
10
11
12
13
14
15
16
5’-Fls-tptp tptptp tptptp tptptp tptpt-3’
6'-ATA AAC AGA GGG ATT TAC Ca-3'
6'-GAG TCA TTC GAC TTC TGA Ct-3’
6’-ATG ATG AGC TAC TGT ATC GCTa-3’
−
−
6439.9657
6364.9085
7061.0162
6439.978
6364.921
7061.030
155.4 (946.0)
151.8 (1023.7)
** Uppercase, underline: TMO; lowercase: 2’-deoxynucleotide pS; lowercase with subscript p: 2’-deoxynucleotide phosphate; Fls:
Fluorescein pS. *Unless specified with subscript p (phosphates), all internucleotide linkages are phosphorothioate (pS) or
thiophosphoramidate. ǂFor HRMS data of TMOs, please see SI, p.S49-S62. For percentage yields (DMT-ON), see SI Table S1, p. S21.
was monitored by LCMS. Under the conditions used for
conventional DMT-On/Off purification34 (80% aqueous
acetic acid), TMO1 was completely degraded within 10 min
at 25 oC(data not shown). Therefore, the acid concentration
was reduced to 50%. The rate of degradation of TMO1 over
time under this condition is shown in SI Figures S15a-e. We
determined that a 5 min treatment with 50% aqueous acetic
acid followed by immediate quenching with triethylamine
resulted in > 99% detritylation of TMO1 (SI Figure S15b)
with minimal degradation, making this our preferred
method for DMT deprotection (See SI Section 3 for details).
Using this protocol, the final DMT-Off oligonucleotides were
> 85 % pure on average as determined by LCMS.
profiles of mixmer ONs revealed that the 6’-DMT ON
product was obtained in 30-45% yield (SI Table S1). We
noted that apart from % GC content and ON length, the
purity
and
moisture
content
of
morpholino
phosphorodiamidites play
a
vital role in obtaining
satisfactory yields. Thus, the average stepwise yields varied
from 95-97% per coupling (ABI-394, trityl monitoring).
After DMT-On/Off purification, the TMO oligonucleotides
were obtained in moderate yields (47-276 nanomoles,
Table 1). Comparable yields were also obtained for TMO-
DNA-pS chimeras (ONs 4, 5, 6; Table 1 and SI Figures S4-S6).
For purposes of yield comparison, 14-mer DNA-pS ON12
and unmodified DNA ON13 produced > 800 nmoles of pure
product under the newly optimized TMO synthesis
conditions. TMOs 8, 10, and 11 with a 3’-morpholino moiety
were synthesized using Universal Support III (Glen
Research, VA). In this case, the first morpholino
phosphorodiamidite coupling step was followed by
oxidation instead of sulfurization (USIII to S2, Figure 2). All
subsequent synthesis steps were identical while using
either solid support. Oligonucleotide cleavage from USIII
support was carried out using 2M ammonia in methanol for
1 h followed by deprotection using 28% aqueous ammonia
at 55 oC for 16 h.
Following this procedure, several TMO mixmer ONs and
chimeric TMO-DNA-pS ONs were synthesized and their
LCMS characterization data is provided in Table1. Where
applicable, the percentage yields of full-length DMT-ON
product versus failures were obtained by integrating the UV
profiles of LCMS data and is provided in SI Table S1, p.21. In
order to further establish proof-of-concept for the robust
synthesis of TMOs using the solid-phase synthesis
procedure as outlined in Scheme 2, the spectra of crude,
unpurified reaction mixtures for TMOs 14-16 are shown in
the SI Figures S12(a), S13, and S14. Integration of UV
5
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Page 6 of 25
3
6’-Fls-TT TTT TTT TTT TTT-3’
x10
DAD1 - A:Sig=254.0,4.0 Ref=360.0,100.0 hk-6067-001.d
1
2
2
3
4
1.5
1
5
6
0.5
0
a
t = 0
7
3
x10
DAD1 - A:Sig=254.0,4.0 Ref=360.0,100.0 sv5-6067.d
8
9
2.5
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1.5
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b
t = 23 h
0.5
0
9
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Response Units vs. Acquisition Time (min)
1
x10
- Scan (rt: 30.379-31.993 min, 79 scans) hk-6067-02.d
-3
-6
-5
8
6
4
2
-4
-2
c
t = 0
0
1
x10
- Scan (rt: 30.552-32.063 min, 74 scans) sv5-6067.d
8
6
4
2
0
t = 23 h
d
600
700
800
900
1000
1100
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1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
2500
2600
2700
2800
2900
3000
Counts (%) vs. Mass-to-Charge (m/z)
Figure 3 LCMS Analysis of TMO8 During the SVPDE Assay. Analytical RP-HPLC Fractions corresponding to TMO8 were collected at
various time points during SVPDE treatment (SI Figures S18-S23: Collected fractions with Rt = 13-16 min). Figure 3(a, b) show the
LCMS UV profiles corresponding to TMO8 after SVPDE treatment (t = 0, 23 h). Figure 3(c, d) compare the m/z data at t=0 (untreated)
and t =23 h.
Enzymatic Stability
products but the remaining minor peaks are from the
enzyme as shown in Figure S16). When subjected to the
same assay conditions, considerable peak broadening was
observed for ON12 within 7 h (SI Figures S26- S29).
Additionally, the appearance of a fast eluting peak (Rt = 1.5
min, Figure S29), presumably arising from 5’-pS-thymidine
mononucleotide, was also observed during this time frame.
While phosphorothioate ONs are more stable towards
enzymatic degradation when compared to unmodified
DNA,35 no enzyme is known to degrade non-ionic PMOs.36
Since TMOs are hybrid molecules containing both
morpholino nucleosides and pS linkages, we hypothesized
that they might possess superior enzymatic stability to
nucleases relative to unmodified DNA.
Undegraded TMO8 and its potential enzymatic
degradation products (n-1, n-2 etc.) might co-elute on a
reverse-phase C18 column during analytical RP-HPLC. To
address this, the peaks observed at each time point during
the SVPDE assay was collected, pooled and reanalyzed by
LCMS. In case of TMO8, the analytical HPLC fractions
corresponding to the UV signal at Rt = 13-16 min at various
time points during the SVPDE assay (SI, Figures S18-S23:
time points 1, 4, 8, and 23 h) were re-analyzed by LCMS
(Figure 3 a, b and SI Figure S24). By comparing these
reanalysis profiles with that of untreated TMO8 and
analyzing their corresponding m/z data (Figure 3c, d: t = 0
and 23 h), we confirmed that TMO8 did not degrade within
23 h during our SVPDE assay conditions. We therefore
concluded that in comparison to DNA-pS ONs, TMOs are
highly stable towards Snake Venom Phosphodiesterase I.
The 3’-exonuclease susceptibility of exclusively
thiophosphoramidate modified TMO8 and DNA-pS control
ON12 (Table 1) were evaluated using Snake Venom
Phosphodiesterase I (SVPDE). While performing enzymatic
hydrolysis experiments, TMO8 and ON12 (13.3 µM) were
incubated at 37 oC in a reaction mixture containing 100 mM
Tris-HCl buffer (pH 8.5), 14 mM MgCl2, 72 mM NaCl and
SVPDE enzyme (0.1 U/ mL). Aliquots (45 µL) were
withdrawn at various time points, heat inactivated and
stored in dry ice until analyzed by analytical RP-HPLC.
Based on analytical RP-HPLC analysis (SI, Figures S18-
S23), no detectable degradation or peak broadening was
observed for TMO8 over a period of 23 h. The undegraded
TMO8, with a Rt = 13-16 min, is the major product (two
minor products at 11 and 11.8 min could be degradation
6
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Journal of the American Chemical Society
The SVPDE assays for DNA-pS ON12 and unmodified
runs for all duplexes are provided in SI Section 6, Figure
S33, pp. S39−S41.
1
2
3
4
5
6
7
8
phosphate DNA (ON13) was performed using the same
protocol as described for TMO8. The fractions
corresponding to the broad peak at Rt = 14−16 min
(Analytical RP-HPLC profiles in SI Figures S26-S29) were
re-analyzed by LCMS (SI Figure S30). Although accurate
quantification of each degradation product could not be
obtained due to partially overlapping signals, the LCMS
analysis of ON12 at t =7 h after SVPDE treatment showed
that essentially complete degradation had occurred. In
comparison, unmodified DNA ON13 was completely
degraded within 15 minutes under these conditions (SI
Figure S31).
When compared to the canonical DNA/DNA duplex, the
TMO3/DNA duplex showed a depression of -12.6 oC (- 0.6 oC
per TMO modification). This loss of binding affinity is
greater than that for the DNA-pS/DNA duplex (Tm
depression of 8.2 oC; -0.4 oC per modification). In
comparison, fluorescently labeled Fl-PMO/DNA exhibited a
Tm close to the native DNA/DNA duplex with a loss of only
1.5 oC − a reduction that could be attributed to the presence
of a 3’-fluorescein label on the PMO. TMO-DNA-pS chimera
4 (32% TMO modified with a gapmer37 configuration, 7
TMO linkages, Table 1), exhibited a depression of -6.6 oC (-
9
10
11
12
13
14
15
16
17
18
19
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22
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49
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55
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59
60
o
We routinely employ 1.0 µM succinyl CPG loaded with 2’-
deoxynucleosides or 2’-OMe nucleosides in order to
generate TMOs (e.g., all ONs in Table 1 except 8, 10 and 11).
Therefore, the 3’-exonuclease susceptibility of TMO7
(Table 1) containing a 2’-deoxythymidine moiety as the 3’-
unit was also evaluated using the above method (100 mM
Tris pH 9, 14 mM MgCl2, 72 mM NaCl, 13.3 µM TMO7, 2x10-
1 U/mL SVPDE enzyme, 37 oC). As expected, no degradation
of TMO7 was observed over a period of 23 h (confirmed by
LCMS re-analysis, SI Figure S32).
0.9 C per TMO modification). Surprisingly, TMOs 5 and 6
which are composed of alternating TMO and DNA-pS
o
linkages presented an increase in Tm by ~ +6.5 C (+0.6 oC
per
TMO
modification)
when
duplexed
with
complementary DNA. To summarize, Tm values for
ON/DNA duplexes of ONs 3-6 displayed the trend:
TMO5/DNA ~ TMO6/DNA> DNA/DNA ~ Fl-PMO/DNA >
TMO4/DNA > DNA-pS/DNA > TMO3/DNA.
Contrary to its DNA duplex, the TMO3/RNA duplex
showed an increase in Tm by +10 oC (+0.48 oC /TMO
linkage) when compared to the unmodified DNA/RNA
duplex. Similarly, Fl-PMO/RNA duplex also showed a Tm
increase of +0.4 oC per modification. TMO5 and TMO6 which
contain alternating morpholino thiophosphoramidate and
Thermal Denaturation Studies
The binding affinity of an oligonucleotide plays a critical
role in shaping its therapeutic potential. Since the advent of
LNA,37 several conformationally locked sugar modifications
have been developed to improve the binding affinity of
ONs.38 Among unnatural backbone constructs, PMOs and
PNAs improve RNA hybridization affinity39-40 whereas
stereorandom ONs consisting of phosphorothioates41 and
boranophosphates42 decrease binding affinity. Although
phosphoramidate ONs23 and chimeric morpholino
phosphoramidate-DNA have exhibited decreased RNA
binding affinity,21 N3’-P5’ phosphoramidates and their
thiophosphoramidate variants as well as triazole-linked
morpholino oligonucleotides20 have been shown to form
highly stable duplexes with RNA.43
o
DNA-pS linkages showed a Tm increase ≥ +9.6 C (+0.88 −
1oC /TMO linkage), which is the highest Tm increase
observed for any duplex discussed in this work.
The DNA-pS/RNA control duplex showed a -0.4 oC
depression per DNA-pS linkage as expected,44 and the
o
gapmer TMO4/RNA duplex showed a depression of -0.5 C
per TMO linkage. This may be attributed to the nature of the
gapmer design (small TMO ‘wings’ and a 14-mer DNA-pS
‘gap’). Thus, the RNA duplexes of TMOs 3-6 followed the
trend: TMO5/RNA ~ TMO3/RNA ~ TMO6/RNA > Fl-
PMO/RNA > DNA/RNA > TMO4/RNA > DNA-pS/RNA.
Encouraged by the increased RNA binding affinity of
various morpholino and thiophosphoramidate analogues,
the hybridization affinity of TMO and TMO-DNA-pS
chimeras 3−6 (Table 1) towards complementary DNA and
RNA were investigated. Control duplexes of the same
sequence consisting of PMO, DNA-pS or canonical DNA were
also evaluated under identical conditions. Briefly, ONs 3-6
and their DNA or RNA complements (1.0 µM in each strand)
were dissolved in a suitable buffer (50 mM Tris-HCl, 50 mM
KCl and 1 mM MgCl2, pH 8.3). DNA and RNA duplexes of
PMO, DNA-pS, canonical DNA were also prepared under
identical conditions and used as controls. Each duplex (total
volume =1.0 mL) was added to a 10 mm path length cuvette
and placed in a 6x6 Peltier thermostatted multi-cell holder.
The samples were heated from 15−90 oC at a 3 oC/min ramp
rate, maintained at 90 oC for 5 min and cooled to 10 oC at a
To summarize, alternately modified TMO5 and TMO6
o
showed an increase in binding affinity (+6−10 C) towards
both complementary DNA and RNA. We hypothesize that
the insertion of alternating 2’-deoxynucleotides after each
TMO subunit might provide increased flexibility to an
otherwise rigid construct, permitting more efficient binding
with the complementary strand.
A significant loss Tm (>12 oC) was observed for the
TMO3/DNA duplex. Similarly, Fl-PMO also did not show
increased binding affinity towards complementary DNA,
suggesting that the exclusive presence of rigid morpholino
moieties results in a backbone conformation that is not
conducive to efficient DNA binding. The DNA-pS/DNA
duplex also showed a reduced Tm (-8.2 oC), as expected.
Thus, it can be hypothesized that the TMO3 construct, being
a perfect hybrid of both rigid morpholino sugars and pS
internucleotide linkages, suffers a cumulative loss of DNA
binding ability. In contrast, both TMO3 and Fl-PMO showed
superior RNA binding affinity (+8−10 oC).
1
oC/min ramp rate before initiating thermal runs (0.5
oC/min ramp rate). Each experiment was repeated at least
two times per duplex (4 curves). Representative thermal
7
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Journal of the American Chemical Society
Table 2. Tm data of DNA and RNA duplexes of TMOs 3-6
Page 8 of 25
1
Sequence Design#,ǂ
2
3
Duplex*
TMO
Mod
Tm
SD
∆Tm
∆Tm/
mod¶
4
5
DNA/DNA
5’-tp gptpap apapcp cpaptp gpaptp gptpgp cptpgp cptpa-3’
0
64.82
0.4
−
−
6
7
8
9
DNA-pS/DNA
TMO3/DNA
TMO4/DNA
TMO5/DNA
TMO6/DNA
Fl-PMO/DNA
DNA/RNA
5’-t gta aac cat gat gtg ctg cta-3’
0
56.60
52.20
58.21
71.41
71.77
63.32
64.45
-8.22
-12.63
-6.61
6.59
6.95
-1.50
−
-0.40
-0.60
-0.90
0.66
0.63
0.0
0.3
1.5
0.6
0.9
0.5
0.7
0.1
6’-T GTA AAC CAT GAT GTG CTG CTa-3’
6’-T GTA aac cat gat gtg ctG CTa-3’
5’-tGtA aAc CaT gAt GtG cTg Cta-3’
6’-T gTaA aCc AtG aTg TgC tGc Tat-3’
6’-t gta aac cat gat gtg ctg cta-Fl-3’
5’-tp gptpap apapcp cpaptp gpaptp gptpgp cptpgp cptpa-3’
21
7
10
11
12
13
14
15
16
17
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21
22
23
24
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37
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10
11
0
0
−
DNA-pS/RNA
TMO3/RNA
TMO4/RNA
TMO5/RNA
TMO6/RNA
Fl-PMO/RNA
5’-t gta aac cat gat gtg ctg cta-3’
0
56.20
74.43
60.68
74.50
74.10
72.65
-8.25
9.98
-0.40
0.48
-0.54
1.00
0.88
0.40
0.4
0.2
0.4
0.9
0.7
0.6
6’-T GTA AAC CAT GAT GTG CTG CTa-3’
6’-T GTA aac cat gat gtg ctG CTa-3’
5’-tGtA aAc CaT gAt GtG cTg Cta-3’
6’-T gTaA aCc AtG aTg TgC tGc Tat-3’
6’-t gta aac cat gat gtg ctg cta-Fl-3’
21
7
-3.77
10.05
9.65
10
11
0
8.19
*Canonical DNA, Fl-PMO and DNA-pS duplexes were utilized as controls. # Complementary strands were unmodified DNA or RNA;
ǂUppercase, underline: TMO; lowercase: 2’-deoxynucleotide pS; lowercase, subscript p: 2’-deoxynucleotide phosphate; lowercase,
italics: PMO; Fl: Fluorescein. ¶ no TMO modifications are present in Fl-PMO: Tm/mod was calculated based on Tm difference w.r.t
the corresponding DNA/DNA or DNA/RNA control duplex. SD: standard deviation.
Apart from the percent modification, the position of TMO
linkages within the sequence appears to play a crucial role
in determining its binding affinity. This becomes obvious
while comparing the Tm values of TMO5, TMO6 (50% TMO
modified) and gapmer TMO4 (32% TMO modified). When
complexed to complementary RNA or DNA, the binding
affinity of the latter was lower by ~ 14 oC.
intensity ~210 nm and a positive band of strong intensity
~270 nm.48 The positive Cotton band and the negative band
at 210 nm characteristic of an A-form helical structure is
most prominent in the TMO5/RNA heteroduplex, and
closely resembles a typical A-form RNA/RNA duplex shown
in SI Figure S34.49 This observation further supports the
hypothesis that the increased conformational flexibility
gained by inserting 2’-deoxynucleotide linkages between
Circular Dichroism Spectra
TMO subunits imposes
conformation in its duplexes resulting in superior binding
affinity for this design.
a more pronounced A-form
The conformation of canonical DNA/RNA heteroduplexes
lies between A and B form helical geometries.45 While both
A and B form duplexes have maxima and minima at similar
wavelengths, greater ellipticities between 270 nm and 280
nm is observed in the A-form, whereas a B-form duplex has
approximately equal positive and negative bands above 220
nm with a crossover at 261 nm.46 To gain additional insights
into their global helical structure, RNA heteroduplexes of
TMOs 3, 5 and 14 were assessed using circular dichroism
and compared with control duplexes (DNA-pS/RNA,
canonical DNA/RNA). Since the relative position and
intensities of characteristic negative bands at 210 nm and
the positive band around 280 nm for DNA/RNA hybrids are
highly sequence dependent,47 TMOs 3-6 and a DNA-pS
control of identical sequence were used during this
experiment, with the exception of TMO14 (Table 1).
Crossover bands between 205 to 215 nm and 227 to 233 nm
were observed for all duplexes except DNA-pS/RNA, which
showed no crossover in these regions. The positive
absorption band at 273 nm for the canonical DNA/RNA
duplex (crossover at 257 nm) was blue shifted to ̴265 nm
for all TMO duplexes as well as the DNA-pS/RNA duplex
(crossover wavelengths between 250-255 nm). Apart from
the TMO5/RNA duplex, the gapmer TMO4/RNA duplex and
the DNA-pS/RNA duplex also exhibits an intense negative
peak in the 200-213 nm range which is characteristic of an
A-form duplex.48 In case of TMO14, this negative peak
closely resembles that of canonical DNA/RNA control
duplex (both duplexes crossover at 203, 211 nm). However,
a lesser intense negative peak was observed for TMO3/RNA
duplex which highlights the sequence dependence effects
observed in CD spectra. In summary, the CD spectra of
As seen in Figure 4, the CD spectra of RNA duplexes of
TMOs 3, 5 and 14 display the characteristic features of an A-
type helical environment with negative bands of varying
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1
2
3
4
5
6
7
8
9
15
10
5
TMO3/RNA
TMO4/RNA
DNA-pS/RNA
DNA/RNA
TMO14/RNA
DNA/DNA
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
TMO5/RNA
0
-5
-10
205 215 225 235 245 255 265 275 285 295 305 315 325
Wavelength
Figure 4 Circular Dichroism of TMOs 3, 4, 5 and 14 relative to DNA-pS/RNA, DNA/RNA and DNA/DNA as controls. An example for
an A-form RNA/RNA control duplex is provided in SI Figure S34.
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
newly synthesized TMO heteroduplexes reveals its
structural similarities to the DNA-pS/RNA duplex.50 TMOs
3, 4 and 14 adopt helical conformations which are closer to
an A-form rather than a B-form duplex. Interestingly, the
uniformly TMO modified constructs (TMO3 and 14) yield
RNA duplexes with notably different biophysical
characteristics relative to chimeric TMO5. Thus, the
TMO5/RNA duplex closely resembles an A-form RNA/RNA
duplex, providing a logical explanation for its higher
binding affinity towards complementary DNA and RNA.
Further experiments designed to evaluate the helical
conformations adopted by various TMO/DNA duplexes are
currently underway.
retroviral reverse transcriptases (RT) contain an RNase H
domain which is essential for reverse transcription of viral
cDNA, the RNase H domain of HIV-RT is being evaluated as
a potential target for the treatment of drug resistant HIV-1
strains.53
Antisense-based RNase H therapeutics gained substantial
interest over four decades ago when Zamecnik et al.
demonstrated that addition of short synthetic ONs (13-
mers) targeting the viral repeat sequence of Rous sarcoma
virus inhibited viral replication and oncogenic
transformation of chicken fibroblast cells.54 They
successfully demonstrated the ability of ONs to inhibit gene
expression by binding and then degrading the RNA target
through the action of RNase H1.55 Since this ground
RNase H1 Activity of TMO3/RNA and Gapmer
TMO4/RNA Duplexes
breaking
work,
several
chemically
synthesized
oligonucleotide analogues have been evaluated for RNase
H1 mediated antisense therapies. This is an attractive
strategy since the reaction proceeds catalytically by
releasing the ON drug for several successive rounds of RNA
targeted inhibition. Examples of RNase H1 active chemical
RNase
H enzymes constitute a large family of
endonucleases that specifically digest the RNA strand which
is hybridized to complementary DNA. These evolutionarily
abundant, ancient enzymes are involved in numerous
biological processes including degradation of RNA primers
from Okazaki fragments during DNA replication,
homologous recombination, DNA repair and RNAi.51-52 Since
modifications
include
phosphorothioates,56
boranophosphates57, mesylphosphoramidates58 and sugar
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modifications such as arabinonucleic acid,59 cyclohexene
RNase H1 activity at a previously characterized cleavage
site.65-66
Based on these findings, we hypothesize that the lack of
nucleic acid60 and 2’-fluoro arabinonucleic acid.61
1
2
3
4
5
6
7
8
TMOs exhibit higher RNA binding affinity relative to
canonical control duplexes, possibly allowing them to
efficiently invade RNA secondary structures. They also
possess superior enzymatic stability which makes them
ideal candidates for RNase H1 based therapeutics. To
evaluate this aspect, TMO3 was preannealed with a 5’-
fluorescein labeled complementary RNA and digested with
Escherichia coli RNase H1. The results were analyzed by
PAGE (detailed experimental protocol in SI Section 7) and
visualized using a transilluminator to detect the 5’-
fluorescein tag attached to RNA (Lanes 1, 2; Figure 5). No
degraded 5’-Fl-RNA fragments were observed in case of the
TMO/RNA duplex after RNase H1 treatment. We therefore
concluded that the TMO3/RNA duplex is not RNase H1
active.
RNase H1 activity of the TMO3/RNA duplex might be due to
its lack of conformational flexibility due to the presence of
rigid, 6-membered morpholino rings throughout its
backbone, which precludes it from entering the phosphate
binding pocket or interacting effectively with the DNA
binding channel within the enzyme’s catalytic site. This
hypothesis is further supported by the work of ∅stergaard
et al;66 they recently demonstrated that a fluorinated
nucleoside modification containing a rigid six-membered
ring (FHNA), when systematically walked through every
position along the gap region of a 3-9-3 gapmer showed a
marked decrease in RNase H1 activity at most positions
when compared to its 2’-fluoro-2’-deoxynucleoside analog
(FRNA).66
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It is well known that RNase H1 inactive ONs can be
rendered RNase H1 active by adopting a gapmer ON design,
where such modifications flank the RNase H1 active DNA or
DNA-pS segment.62 We therefore explored the RNase H1
activity of gapmer TMO4 containing TMO wings and a 14-
mer DNA-pS gap. The experiment was carried out using the
same protocol as described for TMO3. The results shown in
Figure 5 (Lanes 3, 4) illustrate that TMO4 stimulates RNase
H1 activity.
1
3
4
5
6
2
Further insights into the RNase H1 recruiting ability of
certain chemical modifications versus those that are
inactive can be gained from the crystal structure analyses of
human RNase H1 complexed with several DNA/RNA
duplexes, as described by Nowotny et al.63 Although the
RNA-binding domain of the enzyme directs positional
preference for the cleavage site,64 its catalytic core primarily
interacts with the minor groove of an 11-bp RNA/DNA
duplex segment. The RNA strand precisely fits into one of
the two grooves present in the active site where it adopts a
regular A-form structure and is largely recognized through
interactions with four 2’- hydroxyl residues located around
the scissile phosphorus within the catalytic core.
DNA modifications with high flexibility have been shown
to ablate RNase H1 activity.65 Nevertheless, the specificity of
the DNA strand is imposed by a conformational flexibility
requirement that permits its inclusion into the phosphate
binding pocket. Since large distortions to the torsion angles
of the ON backbone are required to position the DNA strand
within this pocket, only a flexible B-form oligonucleotide
can satisfy this requirement. Additionally, this strand must
be able to precisely fit within the DNA-binding channel
formed by the “basic protrusion” site. It is also required to
undergo numerous physical distortions within this channel,
resulting in a heteroduplex that is between A and B form.
The specificity of DNA over RNA is further imposed by a
steric requirement, i.e., the absence of 2’-hydroxyl groups,
which in turn allows hydrogen bonding and van der Waals
interactions within the basic protrusion channel. Thus,
RNase H1 has highly complex structural requirements such
Figure 5 Gel electrophoresis images of 5’-Fl-RNA duplexes of
TMOs 3, 4 and control DNA-pS following the E. Coli RNase H1
assay. Fluorescence arises only from intact 5’-Fl-RNA or its
degraded fragments. Lane 1: TMO3/RNA duplex ctrl; Lane 2:
TMO3/RNA duplex + RNase H1; Lane 3: TMO4/RNA duplex
ctrl; Lane 4: TMO4/RNA duplex + RNase H1; Lane 5: DNA-
pS/RNA duplex ctrl; Lane 6: DNA-pS/RNA duplex + RNase H1.
TMO4, on the other hand, encompasses a large 14
nucleotide DNA-pS gap, and is RNase H1 active. This can be
explained based on previous work that a 7-10 nucleotide
gap length was sufficient to allow for efficient RNase H1
catalysis67 but a complete loss in activity occurs when the
gap length was < 5 deoxynucleotides.68 Additionally,
comprehensive sequencing-based experiments that
characterized the sequence preferences of various RNase
H1 enzymes have shown that RNA cleavage sites predicted
that even
a
single chemical modification when
appropriately positioned within a DNA strand can abolish
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of miR-15b-5p using anti-miRs derepresses Renilla gene
to have the highest probability for RNase H1 cleavage falls
within this gap length.69
expression. This increased expression of RLuc can be
monitored by measuring the luciferase activity of both RLuc
and FLuc using the Dual Luciferase reporter assay
(Promega). Thus, an increase in the production of RLuc
correlates with the effectiveness of the anti-miR to bind to
endogenous miR-15b, while FLuc provides an internal
control to which experimental values can be normalized.
The dynamic range of reporter assays are proportional to
the copy number of the chosen miRNA target in cells.
Inhibiting more abundant miRNAs (~10,000 copies per
cell) can result in a larger fold change in RLuc expression
compared to miR-15b-5p, which has moderate abundance
(~ 800 copies per cell) in HeLa cells.78
1
2
3
4
5
6
7
8
9
In summary, exclusively TMO modified ONs (e.g., TMO3)
do not elicit RNase H1, presumably due to their
conformational rigidity. Nevertheless, a gapmer design such
as TMO4 is rendered RNase H1 active. This suggests that
exclusively TMO modified ONs are potential candidates for
exon skipping experiments where they function as steric
blockers, whereas gapmers having TMO wings can be used
for antisense experiments that involve mRNA degradation
through activation of the RNase H1 pathway.
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Evaluation of TMOs as miRNA Inhibitors
MicroRNAs (miRNAs) are short non-coding RNAs that
play major roles in post-transcriptional gene regulation by
fine-tuning both the stability and rate of translation of
mRNAs.70 They suppress translation by guiding the miRISC
complex to partially complementary sites in the 3’-UTR of
their mRNA target, resulting in deadenylation, translational
repression or mRNA degradation.71 They have been widely
implicated in the pathogenesis of various human diseases
making them attractive targets for oligotherapeutics.
As a preliminary screen for biological activity, TMOs 3, 4
and 5, designed with varying number and placement of TMO
linkages, were screened in the 0.1-1ꢀμM concentration
range in the HeLa-15b system described above. A non-
targeting control (TMO9) and adequate mock controls
(Oligofectamine only transfection to control for the effect
of transfection reagent on cells) were used. An anti-miR of
identical sequence, but exclusively 2’-OMe modified with
alternating phosphate and phosphorothioate linkages was
tested as positive control. A DNA-pS ON of identical
sequence was used as the negative control. All ONs used in
this study except TMO9 were full-length reverse
complements of endogenous mature miRNA-15b-5p.
Antisense microRNA inhibitors (anti-miRs) are
chemically modified short ON analogues that are
complementary to an endogenous mature miRNA of
interest. They function by competitively sequestering the
miRNA target in the cytoplasm resulting in miRNA
inhibition and consequent derepression of its mRNA target.
Although their mechanism of action is not fully understood,
anti-miRs were originally thought to stimulate miRNA
degradation through an RNase H1-dependent pathway.
However, more recent studies have demonstrated that anti-
To perform the experiment, each ON was transfected via
lipofection to 60% confluent HeLa-15b cells (0.1−1ꢀμM of
each ON per 96-well). After transfection, the cells were
harvested, lysed and analyzed at 24 h. The biological activity
was assayed using the Dual-Luciferase Reporter Assay
System (Promega) according to manufacturer’s
instructions. The ratio of Renilla luminescence to Firefly
luminescence (RLuc/FLuc) for ON-treated cells was
miRs inhibit miRNA function by
a steric blocking
mechanism and physically associate with Argonaute-bound
miRNAs.72 The consistently poor activity of potent gapmer
designs in miRNA assays has led to the hypothesis that the
Argonaute-associated miRNA/anti-miR complex may not
be accessible to RNase H1.73-74 Chemical modifications such
as 2′-OMe, 2′-O-methoxyethyl, 2′-fluoro and LNA have
demonstrated effective miRNA targeting during in vitro or
in vivo experiments.75-76
quantified using
a luminometer and this data was
normalized to the (RLuc/FLuc) ratio of mock-transfected
cells and reported as fold change in RLuc/FLuc with respect
to the lipid-only transfection control, which was set to 1.
Thus, a taller histogram corresponds to higher RLuc
expression due to more efficient miR-15b-5p inhibition. The
results of this experiment are shown in SI Chart S1, p. S44.
Luciferase reporter gene assays are commonly employed
to evaluate anti-miR potency in miRNA-mediated gene
regulation. To investigate whether TMOs can function as
miRNA inhibitors in vitro, we used a HeLa cell line with two
stably integrated luciferase reporters.77 The Firefly
luciferase gene (FLuc), under the transcriptional control of
a weak upstream constitutive promoter and a downstream
poly(A) signal acted as an internal control. A perfectly
complementary binding site for hsa-miR-15b-5p was
inserted between the Renilla luciferase (RLuc) coding
sequence and the poly(A) signal located at the multicloning
site of psiCHECK-2, (Promega) to transcribe a chimeric
Renilla mRNA which contains both the Renilla-coding
sequence and the mir-15b-5p target site (Vector map
provided in SI, Section 9). When the reporter plasmid is
stably expressed in HeLa cells, Renilla luciferase expression
is suppressed by endogenous miR-15b-5p, presumably by
binding with perfect complementarity to its 3’UTR and
subsequently degrading the RLuc mRNA. Potent inhibition
The DNA-pS control showed poor activity throughout this
assay, whereas the chimeric 2’-OMe control and TMO5
showed 6 to 7-fold improved activity within 24 h of
transfection. TMO3 (exclusively TMO modified) showed
moderate levels of activity, resulting in a 3 to 4-fold change
in RLuc expression. Gapmer TMO4 did not show
appreciable levels of activity. The non-targeting control
TMO9 showed a roughly 2-fold increase in the 0.25−1 µM
concentration range implying the presence of off-target
effects at high concentrations. The maximum fold-change of
RLuc expression was observed for the 2’-OMe control,
followed by TMO5 (6 to 7-fold change), and no further
increase in activity was observed when the concentrations
were increased beyond 100 nM. This could be due to the low
to moderate copy numbers of miR-15b-5p in HeLa cells,78
implying that maximum inhibition might have been
achieved at 100 nM. Although a dose-dependent inhibition
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1
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7
8
14.0
12.0
10.0
8.0
48 h
72h
Seed Region
hsa-miR-15b-5p: 5’-UAG CAG CAC AUCAUGGUUUACA-3’
2’-OMe control: 3’-A*UC*GU*CG*UG*UA*GU*AC*CA*AA*UG*U -5’
anti-miR TMO3: 3’-aTC GTC GTG TAG TAC CAA ATG T-6’
anti-miR TMO4: 3’-a TCG tcg tgt agt acc aaA TGT-6’
anti-miR TMO5: 3’-a tCg TcG tGt AgT aCc AaA tGt-5’
anti-miR TMO6: 3’-ta TcG tCg TgT aGt AcC aAa TgT-6’
DNA-pS control: 3’-a tcg tcg tgt agt acc aaa tgt-5’
9.6
7.8
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6.0
5.6
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4.0
3.1
3.0
2.8
2.2
2.2
2.0
1.6
1.6
1.4
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1.3
1.2
1.2
1.2
1.2
1.1
1.1
0.9 0.9
0.0
DNA-pS
TMO5
TMO6
2'-OMe-ctrl
TMO3
TMO4
TMO9
Chart 1 In vitro evaluation of TMOs 3-6 as miR-15b-5p inhibitors. Dose-dependent derepression of RLuc mRNA was
observed for TMOs 5 and 6. Anti-miR TMOs 3-6 directed against miR-15b were designed with varying the number
and placement of TMO linkages and transfected at concentrations ranging from 10 to 100 nmol/L into HeLa-15b cells
stably expressing a psiCHECK-based dual reporter plasmid. Each datapoint represents the mean of four biological
replicates per concentration per time point ±SD. Italics: RNA; Uppercase, bold: 2’-OMe sugar; ‘*’ represents position
of pS linkages; Uppercase, bold, underline: TMO; lowercase: 2’-deoxynucleotide pS.
could not be observed at higher concentrations, this
experiment demonstrated that TMO5 is ~6 fold more active
compared to the DNA-pS (negative) control.
increased nuclease stability. In comparison, TMO5 and
TMO6 (Tm = 74 C, Table 2) consisting of alternating TMO
o
and DNA-pS linkages also produced a 7-fold change in RLuc
expression at 100 nM concentration. Despite its high RNA
binding affinity, the exclusively TMO modified anti-miR
construct (TMO3, Tm = 74.4 oC, Table 2) was less active and
showed only a three-fold inhibition relative to mock-
transfected cells. Although the exact reasons remain
unclear, this could be attributed to its inability to invade and
bind the miRISC complex, poor transfection efficiency or
altered intracellular distribution relative to TMO5 and
In summary, preliminary screening at high ON
concentrations indicated that one or more TMO designs
may have the potential to be further investigated as anti-
miRs. However, it is well known that cellular toxicity, anti-
proliferative effects and the manifestation of off-target
effects by non-specific binding increases with increasing ON
concentration. We therefore attempted to identify the
lowest effective doses for the above TMO designs by
performing this experiment at a lower concentration range
(0.1−100 nM). The transfection protocols were carried out
as described earlier.
o
TMO6. Although the gapmer anti-miR TMO4 (Tm = 60.7 C,
Table 2) with TMO wings and a 14-mer DNA-pS gap is
capable of eliciting RNase H1 activity, it was not a potent
inhibitor miR-15b-5p, presumably due to its poor target
binding ability and intrinsic susceptibility to endonucleases.
The DNA-pS anti-miR (negative control) showed poor
activity at all concentrations tested. This is can be explained
based on its reduced RNA binding affinity (Tm= 56.2oC,
Table 2). The 2’-OMe positive control containing 50 %
As shown in Chart 1, the highest miRNA inhibition levels
were achieved at 100 nM. A maximal fold-response (~ 8-
fold) was detected for TMO5 at 100 nM concentration, 48 h
time point. No further increase in activity was observed
either at higher TMO5 concentrations (SI Chart S1), or at
longer time points for either the TMO-based designs or the
2’-OMe positive control. This is presumably because the
dynamic range of the luciferase assay is limited by the
moderate copy numbers of miR-15b-5p in HeLa cells.78
phosphorothioate linkages (predicted Tm
=
77.2 oC)
effectively inhibited mir-15b-5p at 100 nM concentration;
greater than 7-fold change in RLuc expression was
observed. This is because the increased binding affinity of
2’-OMe sugars (22-mer) compensates for the decrease in Tm
caused by 11 pS linkages (~ -0.5 to -0.7 oC/mod), resulting
in a biologically active anti-miR with high target affinity and
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Anti-miR activities observed at 48h were sustained at
complements. Additionally, it appeared that the placement
of rigid morpholino sugars uniformly throughout the ON
backbone results in a relatively rigid construct that binds
with high affinity to RNA, but not DNA. This lack of flexibility
is also suggested by the observation that an exclusively
TMO modified ON was RNase H1 inactive. Based on CD
spectra, the global helical structure of all TMO/RNA
heteroduplexes lies between A and B forms, although the
alternately TMO modified chimera displayed prominent A-
form bands, approaching a typical RNA/RNA duplex.
Preliminary bioactivity screens that test the potency of
various TMO designs as anti-miRs showed that some TMO
designs may find applications as microRNA inhibitors. In
summary, we have demonstrated that TMOs show great
promise to be utilized as effective research tools in
molecular biology and may have significant potential as
novel drug candidates in oligotherapeutics. Efforts are
currently underway to further expand the scope of TMO
based drug candidates in various therapeutic modalities
and elucidate their mechanism of action.
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5
6
7
8
72h, suggesting that TMOs 5 and 6 have sufficient in vitro
stability over this time period. Although the 2’-OMe positive
control displayed the most potent inhibition in the 25−50
nM range, its activity dropped below that of TMOs 5 and 6
at the lowest tested concentration (10 nM). The higher
potency of TMOs 5 and 6 at 10 nM might be due to their
enhanced nuclease stability, resulting in the availability of
more undegraded anti-miRs over a longer time period.
Additionally,
a dose-dependent increase in RLuc
9
expression was observed for alternately modified TMOs 5,
6 and the 2’-OMe control in the 10-100 nM concentration
range; between 2 to 5-fold increase in RLuc levels was
observed at 10 nM, but more robust RLuc derepression
resulted in a 7 to 8-fold inhibition at 100 nM. The DNA-pS
control and gapmer TMO4 did not effectively inhibit mir-
15b-5p and showed negligible activity during this assay.
The non-targeting control TMO9 maintained similar activity
levels as the DNA-pS negative control in the 10−100 nM
concentration range, suggesting that higher sequence
specific binding and fewer off target binding might occur
under these conditions.
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ASSOCIATED CONTENT
In summary, suitably designed TMOs can effectively
function as miRNA-15b-5p inhibitors in vitro. A dose
response study in the 10-100 nM range showed that higher
levels of derepression of RLuc mRNA can be achieved with
increasing concentrations of TMOs. This experiment further
demonstrated that the number and distribution pattern of
TMO linkages play a critical role in determining biological
activity. This is in agreement with several literature reports
which have shown that the placement of high affinity
modifications such as 2’-F, 2’-OMe, 2’-MOE, LNA or a non-
nucleotide modifier (ZEN linker) wholly dictates the
potency of the resulting ON to modulate the activity of miR-
21.74,79 Further experiments are underway to elucidate both
the in vitro and in vivo potency of various TMO designs in
suitable disease models.
Supporting Information. Procedures for the chemical
1
synthesis of 6′-O-DMT-phosphorodiamidites and their H, 13C,
31P NMR, HRMS data, LCMS characterization of TMO
oligonucleotides, analytical RP-HPLC profiles for SVPDE assay,
Tm curves, Dual-Luciferase assay data. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
krishna.heera@colorado.edu
marvin.caruthers@colorado.edu
Present Addresses
Conclusions
†Centre of Molecular and Macromolecular Studies, Polish
Academy of Sciences, Department of Bioorganic Chemistry,
Sienkiewicza 112, 90-363 Łódź, Poland.
In conclusion, we have successfully developed a chemical
synthesis strategy for preparing a new class of synthetic
molecules called thiophosphoramidate morpholinos using
phosphoramidite chemistry. In contrast to P(V)-based PMO
synthesis, the phosphoramidite-based approach lends itself
to the easy incorporation of several valuable antisense
modifications such as 2’-OMe, 2’-O-MOE, LNA, and others
that are commercially available, providing an easy synthetic
route to generate an array of previously unexplored drug
Author Contributions
All authors have given approval to the final version of the
manuscript.
Funding Sources
This work was funded by the University of Colorado at
Boulder.
candidates for oligotherapeutics.
A combination of
morpholino nucleobases and phosphorothioate linkages
resulted in an oligonucleotide construct with exceptional
nuclease stability. Based upon a 3’-exonuclease degradation
assay using Snake Venom Phosphodiesterase I, TMOs
showed minimal degradation over 23 h. They were three
times more stable relative to a DNA-pS control. Based on Tm
studies, both the number and placement of TMO
modifications play a crucial role in dictating binding
affinity; while a TMO-DNA-pS gapmer performed poorly,
alternately modified TMO-DNA-pS chimeras displayed
excellent hybridization affinity towards both DNA and RNA
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Dr. Annette Erbse and the Shared Instruments Pool
of the Department of Biochemistry at the University of
Colorado Boulder for the CD spectrometer. We thank Dr.
Thomas Lee and the Mass Spectrometry Core Facility for HRMS
analysis. The HeLa-15b cell line was a kind gift of miRagen
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17.
Summerton, J. E.; Weller, D. D.; Stirchak, E. P. Alpha-
Therapeutics, Inc. We thank Dr. Katie Robinson and Dr.
Christina Dalby for their valuable inputs during the Dual
Luciferase assay development.
morpholino ribonucleoside derivatives and polymers thereof. U.S.
Patent No. 5,235,033. 10 Aug. 1993.
18.
1
2
Caruthers, M.; Paul, S. Synthesis of backbone modified
3
4
5
6
7
8
9
morpholino oligonucleotides and chimeras using phosphoramidite
chemistry. U.S. Patent Application 16/359,776, July 18, 2019.
ABBREVIATIONS
TMO, Thiophosphoramidate Morpholino; RNase H1,
19.
oligomeric compounds. WO 2018/165564 A1, 09 March 2018.
20. Palframan, M. J.; Alharthy, R. D.; Powalowska, P. K.;
Seth, P. P.; Ostergaard, M. Morpholino modified
Ribonuclease
H1;
ON,
DNA;
Oligonucleotide;
PMO, Phosphorodiamidate
DNA-pS,
Phosphorothioate
morpholino; CD, Circular Dichroism; OD, Optical Density Units.
Hayes, C. J., Synthesis of triazole-linked morpholino
oligonucleotides via Cu(I) catalysed cycloaddition. Org Biomol
Chem 2016, 14 (11), 3112-3119.
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DNA-pS:RNA
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Figure 1 Chemical representation of phosphorodiamidate morpholino (PMO) (1a), phosphoramidate
morpholino (1b), thiophosphoramidate morpholino TMO (1c) and TMO-DNA-pS chimeras (1d).
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Scheme 1 Chemical synthesis of phosphorodiamidite synthons 4a-d. Reagents and conditions: (i) NaIO4
(1.2 equiv.), anhydrous methanol; (NH4)2B2O7 (1.2 equiv.), 6 h (ii) NaCNBH3 (2.0 equiv);CH3COOH (2.0
equiv.), 16h (iii) (OCH2CH2CN)P[N(iPr)2]2 (1.2 equiv.), 5-Ethylthio-1H-Tetrazole (0.5 equiv.),CH2Cl2, 30
min.
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Figure 2 Solid-phase Synthesis of TMO and TMO-DNA-pS Chimeras; Reagents: (i) Detritylation: 3%
trichloroacetic acid/CH2Cl2 (ii) Condensation: 0.12M 5-Ethylthio-1H-tetrazole/CH3CN, 600s wait (iii)
Sulfurization: 0.05 M DDTT/pyridine/CH3CN (iv) Capping: Ac2O/pyridine/THF; 1-Methylimidazole/CH3CN (v)
Deprotection: 28% aqueous ammonia, 55 oC, 16 h or (vi) Deprotection: 2M ammonia in methanol, 60 min,
followed by 28 % aqueous ammonia, 55 oC, 16 h. Amidites A, B are color-coded to simplify the cycle.
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Figure 3 LCMS Analysis of TMO8 During the SVPDE Assay. Analytical RP-HPLC Fractions corresponding to
TMO8 were collected at various time points during SVPDE treatment (SI Figures S18-S23: Collected
fractions with Rt = 13-16 min). Figure 3(a, b) show the LCMS UV profiles corresponding to TMO8 after
SVPDE treatment (t = 0, 23 h). Figure 3(c, d) compare the m/z data at t=0 (untreated) and t =23 h.
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Figure 4 Circular Dichroism of TMOs 3, 4, 5 and 14 relative to DNA-pS/RNA, DNA/RNA and DNA/DNA as
controls. An example for an A-form RNA/RNA control duplex is provided in SI Figure S34.
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Figure 5 Gel electrophoresis images of 5’-Fl-RNA duplexes of TMOs 3, 4 and control DNA-pS following the E.
Coli RNase H1 assay. Fluorescence arises only from intact 5’-Fl-RNA or its degraded fragments. Lane 1:
TMO3/RNA duplex ctrl; Lane 2: TMO3/RNA duplex + RNase H1; Lane 3: TMO4/RNA duplex ctrl; Lane 4:
TMO4/RNA duplex + RNase H1; Lane 5: DNA-pS/RNA duplex ctrl; Lane 6: DNA-pS/RNA duplex + RNase H1.
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Chart 1 In vitro evaluation of TMOs 3-6 as miR-15b-5p inhibitors. Dose-dependent derepression of RLuc
mRNA was observed for TMOs 5 and 6. Anti-miR TMOs 3-6 directed against miR-15b were designed with
varying the number and placement of TMO linkages and transfected at concentrations ranging from 10 to
100 nmol/L into HeLa-15b cells stably expressing a psiCHECK-based dual reporter plasmid. Each datapoint
represents the mean of four biological replicates per concentration per time point ±SD. Italics: RNA;
Uppercase, bold: 2’-OMe sugar; ‘*’ represents position of pS linkages; Uppercase, bold, underline: TMO;
lowercase: 2’-deoxynucleotide pS.
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Art Work (Table of Contents )
41x30mm (600 x 600 DPI)
ACS Paragon Plus Environment