Oligonucleotides Containing Aminated 2′-Amino-LNA Nucleotides: Synthesis and Strong Binding to Complementary DNA and RNA

Article abstract of DOI:10.1021/acs.bioconjchem.7b00061

Mono- and diaminated 2′-amino-LNA monomers were synthesized and introduced into oligonucleotides. Each modification imparts significant stabilization of nucleic acid duplexes and triplexes, excellent sequence selectivity, and significant nuclease resistance. Molecular modeling suggested that structural stabilization occurs via intrastrand electrostatic attraction between the protonated amino groups of the aminated 2′-amino-LNA monomers and the host oligonucleotide backbone.

Full text of DOI:10.1021/acs.bioconjchem.7b00061

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Article
Oligonucleotides Containing Aminated 2’-Amino-LNA Nucleotides:
Synthesis and Strong Binding to Complementary DNA and RNA
Chenguang Lou, Simone V Samuelsen, Niels Johan Christensen, Birte Vester, and Jesper Wengel
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00061 • Publication Date (Web): 23 Mar 2017
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Oligonucleotides Containing Aminated 2’-Amino-LNA Nucleotides:
Synthesis and Strong Binding to Complementary DNA and RNA
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Chenguang Lou,† Simone V. Samuelsen,† Niels Johan Christensen,‡ Birte Vester§ and Jesper
Wengel*,†
†Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and
Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
‡Department of Chemistry, Biomolecular Nanoscale Engineering Center, University of
Copenhagen, Thorvaldsensvej 40, Frederiksberg 1871, Denmark.
§Department of Biochemistry and Molecular Biology, University of Southern Denmark,
Campusvej 55, 5230 Odense M, Denmark.
Corresponding author
Prof. Jesper Wengel
Biomolecular Nanoscale Engineering Center,
Department of Physics, Chemistry and Pharmacy,
University of Southern Denmark,
Campusvej 55,
5230 Odense M, Denmark.
Eꢀmail: jwe@sdu.dk; Tel: +45 65502510
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Abstract: Monoꢀ and diaminated 2’ꢀaminoꢀLNA monomers were synthesized and introduced
into oligonucleotides. Each modification imparts significant stabilization of nucleic acid
duplexes and triplexes, excellent sequence selectivity and significant nuclease resistance.
Molecular modelling suggested that structural stabilization occurs via intrastrand electrostatic
attraction between the protonated amino groups of the aminated 2’ꢀaminoꢀLNA monomers
and the host oligonucleotide backbone.
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INTRODUCTION
Considerable efforts are currently invested in the application of artificial nucleic acids within
biochemistry, biology, nanotechnology and medical science.1ꢀ6 Importantly, applications like
oligonucleotide(ON)ꢀbased diagnostics and therapy usually requires chemically modified
ONs to orchestrate improved binding affinity, high selectivity and increased biostability, all
relative to what is offered by unmodified DNA and RNA ONs.7ꢀ9 This demand has inspired
many modifications such as nucleoside surrogates and phosphate backbone derivatives to
achieve those favorable properties.10ꢀ13 In this context, the locked nucleic acids (LNA and 2’ꢀ
aminoꢀLNA, Fig. 1) represent notable examples in which the ribofuranose moieties are
constrained in Northꢀtype (C3’ꢀendo) conformation via an oxymethylene or azamethylene
bridge between the C2’ and C4’ atoms.14ꢀ16
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Figure 1. Structures of LNA, 2’ꢀaminoꢀLNA, ATꢀLNAꢀT, APꢀLNAꢀT and ABꢀLNAꢀT
nucleotide monomers.
Owing at least in part to their polyanionic nature, a main challenge when applying ONs for
diagnostics and therapy is their limited cellular uptake.4,11 One strategy to alleviate this
problem has been to introduce positively charged groups.17ꢀ24 For example, conjugation of an
oligospermine to the 5’ꢀend of an antisense ON or of one strand of an siRNA has been
reported to induce the desired gene silencing activities.25,26 In addition, introduction of
piperazinoꢀmodified and glysinoꢀmodified aminoꢀLNAꢀT nucleosides into bisLNAs enabled
in vitro double strand invasion to an equal or even more significant level as to LNAꢀT.27
In order to combine highꢀaffinity targeting and charge alleviation, we have attached
norspermidine (pKa ~11)28 via 2’ꢀNꢀalkanoyl linkers to the 2’ꢀaminoꢀLNAꢀT monomer
(monomers APꢀLNAꢀT and ABꢀLNAꢀT, Fig. 1). We show below that both of these novel
aminoꢀLNA monomers induce high duplex and triplex stability, favourable mismatch
discrimination and enhanced nuclease resistance. Molecular modelling demonstrated
recurring electrostatic interactions between the protonated norspermidine moiety and the
phosphate backbone, suggesting that backbone charge shielding is a key mechanism of
structure stabilization. In parallel, a monoaminated analogue was also synthesized (ATꢀLNAꢀ
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T, Fig. 1) for comparison.
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RESULTS and DISCUSSION
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Synthesis of ATꢀLNAꢀT, APꢀLNAꢀT and ABꢀLNAꢀT phosphoramidite monomers (9a-9c)
was accomplished in two steps upon direct condensation between DMTrꢀprotected 2’ꢀaminoꢀ
LNAꢀT (7)29 and carboxyꢀfunctionalised precursors 5b, 5c and Fmocꢀ9ꢀaminoꢀ4,7ꢀ
dioxanonanoic acid (6) (Scheme 1). The latter is commercially available, while synthesis of
precursors 5b and 5c followed a route similar to that described by Hujakka et al. with
appropriate alterations in steps iii and v (Scheme 1).30 Thus, the two primary amino groups of
1 were selectively protected using phthalic anhydride in glacial acetic acid to give 2, which
was further reacted with methyl acrylate in a Michael addition reaction or with ethyl 4ꢀ
bromobutyrate in a nucleophilic substitution reaction to provide 3b and 5c, respectively. Full
deprotection on 3b and 3c was performed with conc. hydrochloric acid to afford 4b and 4c,
respectively, with two free primary amino groups and with the desired carboxy function. After
neutralization using sodium hydroxide, subsequent attempts on Fmoc protection of the
primary amino groups of 4b and 4c were unsuccessful following the published synthesis
protocol.30 Inspired by the wellꢀdeveloped NHS ester chemistry on NH2ꢀfunctionalized
oligonucleotides,31ꢀ33 Fmoc protection on 4b and 4c was efficiently achieved using FmocꢀOSu
in a solvent system of acentonitrile and carbonate buffer (7:3) to produce 5b and 5c,
respectively. The intermdiates 3c, 4c and 5c are new compounds, whereas 2, 3b, 4b and 5b
are known compounds.30 2’ꢀAminoꢀLNAꢀT carboxylic groups of precursors 6, 5b and 5c in
the presence of 1ꢀ[bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀb]pyridinium 3ꢀoxid
hexafluorophosphate (HATU) and N,Nꢀdiisopropylethylamine (DIPEA) to provide 8a, 8b and
8c, respectively. These compounds were finally phosphitylated by reaction with 2ꢀcyanoethylꢀ
N,Nꢀdiisopropyl chlorophosphine to give the three desired phosphoramidite building blocks
9a, 9b and 9c.
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Scheme 1. Synthesis of AT-LNA-T, AP-LNA-T and AB-LNA-T phosphoramidites 9a-ca
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aReagents and conditions: (i) phthalic anhydride, glacial acetic acid, reflux, 1 h or 2 h, n.d. or 98%; (ii) methyl
acrylate, RT 4 h, reflux, 4 h, 77% for two steps from 1; (iii) ethyl 4ꢀbromobutyrate, DIPEA, DMF, 70 °C, 168 h,
70%; (iv) conc. HCl, reflux, 24 h, n.d.; (v) 10% NaOH, carbonate buffer/MeCN, FmocꢀOSu, RT, 1 h or 2 h; 94%
for two steps from 3b to 5b; 71% for two steps from 3c to 5c; (vi) Fmocꢀ9ꢀaminoꢀ4,7ꢀdioxanonanoic acid (6),
HATU, DIPEA, DMF, RT, 1 h, 78%; 5b, HATU, DIPEA, DMF, RT, 1 h, 75%; 5c, HATU, DIPEA, DMF, RT, 2
h, 68%; (vii) 2ꢀcyanoethylꢀN,Nꢀdiisopropyl chlorophosphine, DIPEA, DCM, 1 h or 2 h, RT, 9a 61%, 9b 60%, 9c
92%. (n.d. means that the yield was not determined).
As expected, Fmoc protection of the primary amino groups was compatible with standard
solidꢀphase oligonucleotide synthesis,33,34 and phosphoramidite monomers 9a, 9b and 9c were
efficiently incorporated into ONs by soꢀcalled handꢀcouplings35 in stepwise coupling yields
99%, 97% and 95%, respectively. Diethylamine treatment is indispensable before cleavage of
the ONs from solid support to ensure initial removal of the cyanoethyl group thus eliminating
the risk of attack from deprotected free amino groups on acrylonitrile upon treatment with
aqueous ammonia. Interestingly, the standard deprotection and cleavage protocol (28%
aqueous ammonia, 55 °C, 16 h) was not suitable for APꢀLNAꢀTꢀcontaining ONs, as the
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desired ONs were found not to be the major product components after such treatment. For
example, MALDIꢀTOF MS after attempted synthesis of singleꢀmodified ON4 showed a
dominant peak which was ~115 Daltons lower than the calculated molecular weight of ON4.
It is hypothesized that the bis(3ꢀaminopropyl)amino moiety of the APꢀLNAꢀT monomer was
replaced by a primary amine via a reverseꢀMichael addition reaction followed by reꢀaddition
of ammonia. This issue was resolved using a milder deprotection condition (aqueous
ammonia, room temperature, 14 h), leading to the desired ONs as major products.
Reasonably, when an extra methylene unit was introduced to incorporate monomer ABꢀLNAꢀ
T, ON synthesis was fully compatible with standard deprotection and cleavage condition
strongly supporting the proposed reverseꢀMichael addition pathway as described above. After
DMTrꢀON reversedꢀphase HPLC and/or ionꢀexchange HPLC purification, the composition
and purity (>85%) of modified ONs were confirmed by MALDIꢀMS and ionꢀexchange HPLC
analysis, respectively.
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Each monomer (APꢀLNAꢀT, ABꢀLNAꢀT and ATꢀLNAꢀT) was incorporated into a 9ꢀmer
sequence and their binding affinity was evaluated towards complementary DNA and RNA
strands (Table 1). Both single (ON3, ON4 and ON5) and multiple modifications (ON7, ON8
and ON10) were investigated using ONs containing the corresponding DNAꢀT (ON1) and 2’ꢀ
aminoꢀLNAꢀT monomers (ON2, ON6 and ON9) as controls. The endeavour to synthesize
ONs containing three polyꢀaminated monomers (APꢀLNAꢀT or ABꢀLNAꢀT) failed, but
incorporation of two modifications (ON7 and ON8) was successful. When the central DNAꢀT
was substituted by one of the three aminated monomers (ON3, ON4 and ON5), remarkably
increased duplex stabilities were observed in DNA/DNA contexts. Compared to DNAꢀT and
2’ꢀaminoꢀLNAꢀT controls, up to 8.5 °C and 4.5 °C increase in melting temperature was
observed respectively, with the bisaminated monomers APꢀLNAꢀT and ABꢀLNAꢀT
displaying the most significant increases. Except for ON4 and ON5, the most significant
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increases in thermal denaturation temperatures were observed against complementary RNA
which is consistent with previous results for other 2’ꢀaminoꢀLNAꢀT monomers.14
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Incorporation of three ATꢀLNAꢀT nucleosides (ON10) enhanced duplex stability
significantly more than single incorporation (ON3) regardless of the target strand being DNA
or RNA. For APꢀLNAꢀT and ABꢀLNAꢀT it is evident that the position of the monomer plays
a vital role on DNA:DNA duplex stability, with singlyꢀmodified ONs (central, ON4 and
ON5) conferring almost equal duplex stability to that of the corresponding doublyꢀmodified
ones (ON7 and ON8). This latter result, however, represent an exception and after changing
to low salt experimental condition, higher stabilization for two inclusions than for one was
observed. These binding data are consistent with a favorable electrostatic interaction that
becomes less shielded at lower ionic strength (see modeling section below).
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Table 1. Thermal Denaturation Temperatures of Duplexesa
Complementary DNA
Complementary RNA
Sequence
X
Medium Salt
Low salt
17.5
Medium Salt
Low salt
15.5
5’ꢀGTGATATGC
ꢀ
ON1
ON2
ON3
ON4
ON5
ON6
ON7
ON8
ON9
ON10
32.0
29.5
5’ꢀGTGAXATGC
2’ꢀaminoꢀLNAꢀT
ATꢀLNAꢀT
36.0 (+4.0)
39.5 (+7.5)
40.5 (+8.5)
40.5 (+8.5)
36.0 (+4.0)
41.5 (+9.5)
41.0 (+9.0)
41.0 (+9.0)
49.0 (+17.0)
22.5 (+5.0)
26.5 (+9.0)
30.0 (+12.5)
30.5 (+13.0)
22.0 (+ 4.5)
34.0 (+16.5)
34.5 (+17.0)
27.0 (+9.5)
39.0 (+21.5)
38.0 (+8.5)
40.0 (+10.5)
38.5 (+9.0)
38.0 (+8.5)
42.5 (+13.0)
42.0 (+12.5)
42.5 (+13.0)
49.5 (+20.0)
55.5 (+26.0)
23.5 (+8.0)
26.5 (+11.0)
26.5 (+11.0)
27.0 (+11.5)
27.5 (+12.0)
33.0 (+17.5)
35.0 (+19.5)
35.0 (+19.5)
44.0 (+28.5)
APꢀLNAꢀT
ABꢀLNAꢀT
5’ꢀGXGATAXGC
5’ꢀGXGAXAXGC
2’ꢀaminoꢀLNAꢀT
APꢀLNAꢀT
ABꢀLNAꢀT
2’ꢀaminoꢀLNAꢀT
ATꢀLNAꢀT
aTm values (°C) of unmodified and modified (X = 2’ꢀaminoꢀLNAꢀT, ATꢀLNAꢀT, APꢀLNAꢀT or ABꢀLNAꢀT)
DNA/DNA and DNA/RNA duplexes measured as an average of two independent melting temperature
determinations with a deviation ≤ 0.5 °C. Values in brackets are ꢁTm values measured as the difference in Tm
values between modified and unmodified duplexes. The experiments were carried out at pH 7.0 in medium salt
buffer (5.8 mM NaH2PO4/Na2HPO4 buffer, containing 100 mM NaCl and 0.10 mM EDTA) and low salt buffer
(6.7 mM NaH2PO4/Na2HPO4 buffer, containing 0.10 mM EDTA). The increase in melting temperature of 2’ꢀ
aminoꢀLNAꢀT is in general agreement with previous results reported for ON2 and ON9.14
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Protonation of one or more of the primary amino groups seems to induce a crucial effect
for duplex stabilization, and its importance was further emphasized by studying thermal
denaturation temperatures at reduced ionic strength (Table 1). Thus, compared to DNAꢀT and
2’ꢀaminoꢀLNAꢀT, all modified ONs containing monomer(s) ATꢀLNAꢀT, APꢀLNAꢀT and ABꢀ
LNAꢀT showed even stronger affinity towards DNA and RNA complements at the low salt
buffer condition than at medium salt. It should be noted that it is not surprising that 2’ꢀaminoꢀ
LNAꢀT is unable to alleviate the negative influence from the reduced ionic strength, as the
pKa value of the protonated 2’ꢀamino group is only 6.17.36
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The binding specificity was assessed for the three singlyꢀmodified ONs (ON3, ON4 and
ON5) with the nucleobase varied in the complementary strand opposite to the site of
modification (ESI). Relative to the allꢀDNA control ON1, all modified ONs generally
exhibited better mismatch discriminating ability, except against the RNA target with a central
RNAꢀC. The mismatch studies were also extended to ON10 that includes three ATꢀLNAꢀT
monomers, and this study confirmed the satisfactory baseꢀpairing selectivity of this monomer
as ON10 showed similar discriminative ability as ON1 (ESI).
We used molecular modelling (for details, see ESI S7) to investigate the mechanisms of
stabilization of single ATꢀLNAꢀT, APꢀLNAꢀT and ABꢀLNAꢀT monomers inserted into a
DNA:RNA hybrid duplex (i.e. ON3, ON4, ON5 with complementary RNA, see Table 1). The
possible interaction partners of the amine moiety were explored by performing a
conformational search on the 2’ꢀNꢀalkanoyl linkers with amine moieties while keeping the
atoms of the DNA:RNA duplex frozen to the positions in the most representative structure of
the NMR ensemble (PDBꢀID: 1HHW).37 The most favoured conformations found are shown
in Fig 2. Energies and additional parameters are listed for the 10 lowest conformations in
Table S4. Intrastrand hydrogen bonds to phosphate oxygens were found in most
conformations, and the low energy conformations shown in Fig 2 are representative of the
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binding modes found in the 40 kJ/mol energy window searched. The conformational search
did not find interstrand interaction of the type where the protonated amino moieties of the
LNAꢀT modification form hydrogen bonds with opposite strand phosphates. This reflects the
inability of the linkers to bridge the minor groove of the DNA:RNA duplex. However,
another type of interstrand interaction was found as the secondꢀlowest energy conformer (~0.6
kJ/mol higher than the lowest energy conformer) for APꢀLNAꢀT (Fig 2c), with one protonated
primary amine binding to a neighbouring phosphate oxygen and the other towards the O2’ꢀ
atom on the opposite strand.
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Figure 2. Low energy binding modes found in conformational searches on single ATꢀLNAꢀT,
APꢀLNAꢀT and ABꢀLNAꢀT monomers inserted into the DNA:RNA duplex (PDBꢀID:
1HHW).37 a. lowest energy conformer for the ATꢀLNAꢀT monomer; b. lowest energy
conformer for the APꢀLNAꢀT monomer; c. second lowest energy conformer (∆E = 0.6
kJ/mol) for the APꢀLNAꢀT monomer; d. lowest energy conformer for the ABꢀLNAꢀT
monomer. For clarity, only polar hydrogens are displayed.
This binding mode was also found for ABꢀLNAꢀT (ESI, Figure S28b), but at a higher
relative energy (~15 kJ/mol higher than the lowest energy conformer). Despite the substantial
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predicted difference in energy barrier to populating the interstrand interaction mode for ABꢀ
LNAꢀT and APꢀLNAꢀT, the two modifications yield very similar experimental stabilizations
of the singly modified DNA:RNA duplexes (Table 1). This suggests that the primary mode of
duplex stabilization by ATꢀLNAꢀT, APꢀLNAꢀT and ABꢀLNAꢀT is the interaction between the
protonated primary amines and the neighbouring negatively charged phosphates on the same
strand (i.e. Fig 2a, b, d), as recurrently found in the conformational search. This
electrostatic/hydrogen bond interaction may shield the interstrand electrostatic backbone
repulsion, resulting in increased thermal denaturation temperatures.
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The three new monomers were further incorporated into triplexꢀforming oligonucleotides
(TFOs) to evaluate their triplexꢀstabilizing properties towards the wildꢀtype HIV polypurine
tract38 in a 29ꢀmer dsDNA sequence. The studies were performed on singlyꢀ or doublyꢀ
modified TFOs using TFO1, TFO2, TFO6 and TFO10 containing DNAꢀT and/or LNAꢀT
monomers as references (Table 2). Two substitution patterns were studied with a twoꢀbase or
fiveꢀbase gap between the two monomers to discern a spatial effect. At pH 6, the TFOs
containing monomers ATꢀLNAꢀT, APꢀLNAꢀT and ABꢀLNAꢀT all exhibited significantly
higher triplex stability than TFO1 with a ꢁTm value of up to 12.5 ˚C for a single
incorporation. Incorporation of an additional of the amidated monomers led to even greater
increases in thermal denaturation temperature (up to 17.5 ˚C for the twoꢀbase gap and 20.0 ˚C
for the fiveꢀbase gap). It is noteworthy that only one methylene insertion in the linkage
imparts ~2 ˚C increase in triplex melting temperature (from APꢀLNAꢀT to ABꢀLNAꢀT),
which underlines that seemingly minor changes in molecular design can lead to significant
differences in biophysical properties. In general, the thermal stability of the triplexes
decreased drastically when the pH was increased from 6 to 7, probably due to deprotonation
of N3 of cytosine (pKa ≈ 5.5)39ꢀ41 compromising the formation of the stable C+.GC triplet
under neutral pH.42 However, the amidated monomers induced remarkable increases in triplex
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stability also at pH 7 with the order of stability being ABꢀLNAꢀT > APꢀLNAꢀT > ATꢀLNAꢀT
≥ LNAꢀT.
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Table 2. Parallel Triplex Melting Analysisa
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Sequence
TFO1
TFO2
TFO3
TFO4
TFO5
TFO6
TFO7
TFO8
X
Y
Z
pH 6.0
23.0
pH 7.0
n.d.
DNAꢀT
DNAꢀT
DNAꢀT
DNAꢀT
DNAꢀT
DNAꢀT
DNAꢀT
DNAꢀT
DNAꢀT
LNAꢀT
DNAꢀT
DNAꢀT
DNAꢀT
DNAꢀT
DNAꢀT
LNAꢀT
ATꢀLNAꢀT
APꢀLNAꢀT
ABꢀLNAꢀT
DNAꢀT
DNAꢀT
DNAꢀT
DNAꢀT
DNAꢀT
LNAꢀT
31.5 (+8.5)
31.0 (+8.0)
33.0 (+10.0)
35.5 (+12.5)
36.0 (+13.0)
33.5 (+10.5)
36.0 (+13.0)
40.5 (+17.5)
38.5 (+15.5)
37.0 (+14.0)
41.0 (+18.0)
43.5 (+20.0)
12.0
12.0
15.0
15.5
17.0
16.0
17.0
20.0
22.0
23.5
26.0
28.0
ATꢀLNAꢀT
APꢀLNAꢀT
ABꢀLNAꢀT
LNAꢀT
ATꢀLNAꢀT
APꢀLNAꢀT
ABꢀLNAꢀT
LNAꢀT
TFO9
TFO10
TFO11
TFO12
TFO13
ATꢀLNAꢀT
APꢀLNAꢀT
ABꢀLNAꢀT
ATꢀLNAꢀT
APꢀLNAꢀT
ABꢀLNAꢀT
aTm values (˚C) are an average of two independent melting temperatures with deviation no more than 0.5 ˚C.
Values in brackets are ꢁTm = Tm (LNAꢀT, ATꢀLNAꢀT, APꢀLNAꢀT or ABꢀLNAꢀT) ꢀ Tm (DNAꢀT) at pH 6.0. The
experiments were performed in 10 mM sodium cacodylate, 150 mM NaCl and 10 mM MgCl2 under pH 6.0 and
pH 7.0. The concentration of TFO: target DNA was 1.5 µM:1.0 µM. n.d. = not detected.
A 3’ꢀexonuclease assay was used to evaluate the resistance of 32Pꢀlabelled ON1-ON5
towards nucleolytic degradation upon incubation with snake venom phosphordiesterase
(SVPDE). As depicted in Fig. 3, both the allꢀDNA sequence (ON1) and 2’ꢀaminoꢀLNAꢀT
sequence (ON2) were digested rapidly as no trace of corresponding fullꢀlength ONs remained
after 5 min incubation. For ON1, the digestion was completed in 20 min, whereas ON2
indicated some resistance but still being fully degraded during the 60ꢀmin time course.
Retarded mobility was observed when DNAꢀT was replaced with ATꢀLNAꢀT, APꢀLNAꢀT or
ABꢀLNAꢀT, probably due to a combined effect of additional bulkiness and increased
mass/charge ratio derived from protonation of the primary amines (considering charge as a
sum of negative and positive charges). In stark contrast to ON1 and ON2, ON3ꢀON5 all
showed steadfast resistance towards 3’ꢀexonuclytic digestion with no or only little fullꢀ
degradation after 60 min incubation, each displaying a neat major band which persisted over
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the time course of the experiment. Intriguingly, the final products for ON4 and ON5 showed
even slower gel mobility than the corresponding intact 9ꢀmer sequence. That was not the case
for ON1-ON3. To decipher the composition of these products, a parallel 3’ꢀexonuclease assay
was performed on ON3-ON5 (without 5’ꢀ32Pꢀlabelling) and each sample was desalted using
NAPꢀMicro columns. The ensuing MALDIꢀTOF MS analysis showed that the molecular
weights of three final products (ON3*, ON4* and ON5*) were consistent with the
corresponding 6ꢀmer fragments (5’ꢀGTGAXA; see ESI). Apparently, the relative bulkiness
and protonation of the primary amines in ON4ꢀON5 influence so heavily that the full length
GTGAXATGC 9ꢀmers runs even faster than the GTGAXA 6ꢀmers digest products.
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Figure 3. 20% PAGE denaturing gel showing the timeꢀcourse of SVPDEꢀmediated
degradation of 5ʹꢀ32PꢀGTGAXATGC with ON1 (X = T), ON2 (X = 2ʹꢀaminoꢀLNAꢀT), ON3
(X = ATꢀLNAꢀT), ON4 (X = APꢀLNAꢀT) and ON5 (X = ABꢀLNAꢀT) at pH 8.5 and at 21 ˚C
(ESI). Samples were incubated for 5, 20 and 60 min. The negative control (0 min) was taken
before the enzyme was added. The gel was visualized by autoradiography.
Strong stability was independently reported for ONs containing various aminoꢀLNAꢀT and
LNAꢀT derivatives.21,43ꢀ46 Our recent results with a piperazinoꢀmodified aminoꢀLNAꢀT
monomer likewise demonstrated strong resistance to 3’ꢀnucleolytic digestion.22 Such
extraordinary nuclease resistance for ONs containing a single ATꢀLNAꢀT, APꢀLNAꢀT or ABꢀ
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LNAꢀT is in a sharp contrast to LNAꢀT for which a single modification only induce limited
resistance towards exonucleolytic degradation43ꢀ46 wherefore LNAꢀcontaining antisense ONs
typically are constructed as phosphorothioate oligomers. Thus, the aminated 2’ꢀaminoꢀLNAꢀT
derivatives may enable the use of antisense and antigene oligomers based in full or in part on
phosphodiester linkages.
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CONCLUSION
In conclusion, three novel aminated 2’ꢀaminoꢀLNA monomers (ATꢀLNAꢀT, APꢀLNAꢀT and
ABꢀLNAꢀT) nucleosides have been synthesized. The incorporation of these monomers into
oligonucleotides in substitute for DNA or LNA monomers induced significantly increased
duplex and triplex stabilities, enhanced mismatch discriminating ability and robust stability
against 3’ꢀexonucleolytic digestion, with the bisꢀamidated monomers ABꢀLNAꢀT and APꢀ
LNAꢀT generally outperforming the monoꢀaminated monomer ATꢀLNAꢀT. Molecular
modelling indicated that the duplex stabilization occurs via intrastrand electrostatic
interactions between the phosphate group of the oligonucleotide backbone and the protonated
aminoꢀfunctionalities of the novel aminoꢀLNAꢀT analogues. The resulting charge screening is
proposed to contribute to increased denaturation temperatures by reducing oligo backboneꢀ
backbone repulsion. Those favourable properties render the three monomers promising
candidates for the further development of oligonucleotideꢀbased tools for biotechnology and
therapeutic applications. Furthermore, these novel monomers will in future experiments be
explored to answer the important question of relevance of overall net charge on cell
membrane permeability of oligonucleotides.
EXPERIMENTAL PROCEDURES
Experimental procedures and synthetic data can be found in the Supporting Information.
ASSOCIATED CONTENT
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Supporting Information
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The Supporting Information is available free of charge on the ACA Publications website at
DOI:
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Details on synthesis of substituted 2’ꢀaminoꢀLNAꢀT monomers, oligonucleotide
synthesis, purification and analysis (IEꢀHPLC and MAIDIꢀTOF MS), ultraviolet duplex
melting studies, ultraviolet triplex melting studies, nuclease resistance assay, molecular
modelling; copies of representative IEꢀHPLC curves, MAIDIꢀTOF mass spectra, and UVꢀ
melting curves.
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AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: jwe@sdu.dk.
ORCID
Jesper Wengel: 0000ꢀ0001ꢀ9835ꢀ1009
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The VILLUM FONDEN is thanked for funding The Biomolecular Nanoscale Engineering
Center (BioNEC), grant number VKR022710. Joan Hansen, Tina Grubbe Hansen and Lykke
Haastrup Hansen are thanked for technical assistance.
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