UNA (unlocked nucleic acid): A flexible RNA mimic that allows engineering of nucleic acid duplex stability

Article abstract of DOI:10.1016/j.bmc.2009.06.045

UNA (unlocked nucleic acid) monomers are acyclic derivatives of RNA lacking the C2′-C3′-bond of the ribose ring of RNA. Synthesis of phosphoramidite UNA building blocks of the nucleobases adenine, cytosine, guanine, and uracil is described herein together

Full text of DOI:10.1016/j.bmc.2009.06.045

Bioorganic & Medicinal Chemistry 17 (2009) 5420–5425
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
UNA (unlocked nucleic acid): A flexible RNA mimic that allows engineering
of nucleic acid duplex stability
*
Niels Langkjꢀr, Anna Pasternak, Jesper Wengel
Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark
a r t i c l e i n f o
a b s t r a c t
UNA (unlocked nucleic acid) monomers are acyclic derivatives of RNA lacking the C20–C30-bond of the
ribose ring of RNA. Synthesis of phosphoramidite UNA building blocks of the nucleobases adenine, cyto-
sine, guanine, and uracil is described herein together with their incorporation into RNA strands. UNA
monomers additively decrease nucleic acid duplex stability and can be positioned strategically to induce
either lack of discrimination of mismatches, that is, universal base behavior, or increased discrimination
of mismatches, that is, improved hybridization specificity. UNA-modified RNA duplexes are shown to
structurally mimic unmodified RNA duplexes by CD spectroscopy.
Article history:
Received 8 May 2009
Revised 18 June 2009
Accepted 20 June 2009
Available online 27 June 2009
Keywords:
UNA
Unlocked nucleic acid
RNA mimic
Seco-RNA
Ó 2009 Elsevier Ltd. All rights reserved.
Oligonucleotide
1. Introduction
affinity towards a complementary DNA strand,13 but UNA-modi-
fied RNA strands or duplexes were not described. UNA has been ex-
Chemical modification of the natural nucleotide monomers has
been widely explored to engineer nucleic acid binding affinity and
biological activity of synthetic oligonucleotides (ONs).1–4 LNA
(locked nucleic acid)5–9 monomers (Fig. 1) are well known for their
affinity increasing effect, and short LNA-containing probes for their
excellent binding specificity, that is, capability to discriminate
effectively between fully matched and singly mismatched tar-
gets.10 LNA-modified duplexes formed with RNA complementary
strands are of the A-type and structurally very similar to the corre-
sponding unmodified RNA duplexes as has been shown by CD and
NMR spectroscopy.11,12
plored as a constituent in the central DNA segment of gap-mer
antisense oligonucleotides, and compatibility with RNase H recog-
nition and RNA cleavage was reported.14
Both UNA and LNA can be described as RNA analogues. How-
ever, whereas the additional methylene unit linking the O20 and
C40 atoms of LNA monomers locks the furanose ring into a C30-endo
conformation, the acyclic nature of UNA renders this molecule very
flexible. Therefore LNA and UNA can be considered ‘locked RNA’
and ‘unlocked RNA’, respectively. These antipodal structural char-
acteristics make UNA and LNA complementary with respect to ef-
fect on binding affinity towards DNA targets.5–7,13
UNA (unlocked nucleic acid) is an acyclic analogue of RNA in
which the bond between the C20 and C30 atoms of the ribose ring
has been cleaved (Fig. 1). In 1995 we introduced the thymine
UNA monomer as a modification in DNA oligonucleotides together
with a method for synthesis of the corresponding phosphoramidite
derivative.13 Since then, UNA monomers have been incorporated
into DNA-type oligonucleotides using O20-silylated phosphorami-
dites.14 Furthermore UNA-adenylate trimers were synthesized
and shown to be stable against phosphordiesterases.15,16 It should
be noted that seco-RNA has been used as a term to describe various
acyclic forms of RNA and 20,30-seco-RNA as a term to describe what
herein is termed as UNA.13,14 Along with many other acyclic nucle-
otide modifications, UNA was shown to induce decreased binding
We have initiated a program to broadly evaluate the influence
of UNA monomers on hybridization thermodynamics and nucleic
acid duplex structures, and to explore their use within biotechnol-
ogy. Herein we introduce the synthesis of O20-benzoylated UNA
phosphoramidite derivatives of all four standard RNA nucleobases
B
B
B
O
O
O
O
O
O
O
P
OH
O
P
OH
O
P
O
O
O
O
O
O
O
UNA
RNA
LNA
* Corresponding author. Tel.: +45 65502510; fax: +45 66158780.
E-mail addresses: jwe@ifk.sdu.dk, jwe@chem.sdu.dk (J. Wengel).
Figure 1. Structure of UNA (‘unlocked RNA’), RNA, and LNA (‘locked RNA’)
monomers.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.045

N. Langkj
ꢀr et al. / Bioorg. Med. Chem. 17 (2009) 5420–5425
5421
and demonstrate their full compatibility with automated RNA syn-
thesis. We reveal that UNA-modified RNA:RNA duplexes retain A-
type structures and that strategic UNA modification of 21-mer
RNA strands allow predictable decrease of RNA:RNA or RNA:DNA
duplex stabilities and wide-spectrum modulation of hybridization
specificity.
Relative to the corresponding unmodified duplexes, our preli-
minary thermodynamic analysis indicate favorable entropy
changes and unfavorable enthalpy changes for duplex formation
in the presence of one UNA monomer.18 These results may seem
surprising in light of the flexible noncyclic structure of UNA mono-
mers though the examples of entropically disfavored duplex for-
mation have been reported for the conformationally locked LNA
monomers.19,20 However, whereas a favorable enthalpy change
renders the formation of LNA-containing duplexes overall more
energetically favorable than the formation of unmodified duplexes,
our thermodynamic data confirm overall energetically more unfa-
vorable formation for UNA-containing duplexes in line with the de-
creased Tm values observed.
2. Results and discussion
UNA phosphoramidites were synthesized by an optimized ver-
sion of the published procedure13 for synthesis of the thymine
monomer. O50-DMT protected ribonucleosides 1 were treated with
sodium periodate to induce oxidative diol cleavage of the bond
linking the C20 and C30 atoms of the ribose ring. Subsequent reduc-
tion by sodium borohydride yielded the acyclic nucleosides 2.
Selective benzoylation of O20 was optimized relative to the previ-
ously published procedure for the thymine derivative by replacing
the solvent and lowering the reaction temperature (see caption be-
low Scheme 1). O30-Benzoylated by-products were detected by
analytical TLC, but these did not pose any significant problem dur-
ing column chromatographic purification as they were formed in
only minor amounts. The resulting O20-benzoylated acyclic nucleo-
sides 3 were eventually converted into the phosphoramidite build-
ing blocks 4 by standard phosphitylation (Scheme 1).
The phosphoramidites 4 were used on an automated nucleic acid
synthesizer to obtain UNA-modified RNA oligonucleotides ON2–
ON5, ON7 and ON8 (Table 1). Standard conditions for RNA (O20-
TBDMS method) synthesis were used, and the stepwise coupling
yield of unmodified RNA as well as UNA monomers was >99%. Fol-
lowing standard deprotection, purification and work-up, the com-
position of the synthesized ONs was confirmed by MALDI-MS
analysis and the purity (>80%) was confirmed by ion-exchange
HPLC. It is noteworthy that the incorporation of UNA monomers
proved to be fully compatible with RNA synthesis and cleavage con-
ditions, and that no strand cleavage was observed despite the O20-
acyl protecting group applied on the UNA monomers. The latter
observation can be explained by the flexible structure of UNA mono-
mers leaving no structural preorganization of the deprotected 20-OH
group for nucleophilic attack on the O30-phosphate moiety.
We studied the discrimination of mismatches for the singly
UNA-modified RNA strands ON2, ON3, and ON7 against a mis-
match at the central position no. 11 (Table 1;
DTm values caused
by the presence of a mismatch are shown in brackets). With the
mismatches positioned directly opposite to a UNA monomer
(UNA-G in ON2 and UNA-A in ON7) only modest (ON2) or negligi-
ble (ON7) discrimination was observed. These data reflect the rel-
atively weaker base pairing of UNA relative to RNA monomers, but
it should be noted that the data implicate Watson–Crick base pair-
ing for UNA monomers. Mismatch discrimination only slightly less
than for the unmodified RNA strand (ON1) was observed for ON3
with mismatches neighboring the UNA-U monomer in position
10. This demonstrates that the flexibility of a UNA base is only to
a very limited extent transmitted to a neighboring base positioned
at the 30-side of a UNA monomer.
We next determined Tm values of duplexes formed between 21-
mer RNA strands containing two UNA monomers (ON4, ON5, and
ON8) and DNA/RNA targets (Table 1). For the fully matched du-
plexes, decreases in thermal denaturation temperatures of 7–
12 °C per UNA modification were observed. The stability-decreas-
ing effect of a UNA monomer is thus approximately additive for
two incorporations and independent of the nature of the comple-
mentary strand.
ON5 and ON8 are UNA-modified RNA strands having four RNA
monomers positioned between each of the two UNA monomers
and the central RNA-G monomer (‘distal UNA design’), whereas
the two UNA monomers of ON4 are neighboring the central RNA-
G monomer (‘proximity UNA design’). These designs can be used
to modulate hybridization specificity of UNA-modified RNA
strands relative to unmodified reference strands (here ON1 and
ON6). ON4 reveals that lack of mismatch discrimination can be in-
duced by the ‘proximity UNA design’. This design is thus an alter-
native to the so-called universal bases, which in most cases do not
induce such thermally uniform hybridization.21–25 It is likely the
flexibility of the UNA monomers, and the associated decrease in
stability centrally in the duplexes, which renders mismatched pair-
ing at the central RNA-G monomer of no importance for overall du-
plex stability. On the contrary, for the ‘distal UNA-design’,26
improved mismatch discrimination was observed for all mis-
matches against both DNA and RNA complements.17 The observed
improvements in mismatch discrimination are interesting, and we
consider this novel ‘distal UNA design’ an advancement towards
enabling high base pairing specificity in the central segment of
ꢀ20-mer long oligonucleotides. This design originates from the
assumption that the introduction of local flexibility, and thus local
duplex destabilization, may lead to superior ability to sense the
presence of a single mismatch at a predesigned position of an ON
because of what can be described as cooperativity between mis-
matches and sites of low duplex stability.
The influence of UNA monomers on the thermal stability of dif-
ferent duplexes was studied using UV spectroscopy and the duplex
melting temperatures (Tm values) were determined at the maxi-
mum of the first derivative of the thermal denaturation curves (Ta-
ble 1).17 Incorporation of a single UNA monomer into a central
position of 21-mer RNA strands (ON2, ON3, and ON7) induced de-
creases in thermal stability by 5–8 °C for fully complementary du-
plexes towards DNA or RNA complements. The affinity decreasing
effect is slightly less pronounced than for UNA-modified DNA 17-
mers when hybridized towards a DNA complement.13
B
DMTO
O
B
B
DMTO
DMTO
O
O
i
iii
O
P
OBz
NPri2
OH
OH
OH OR
NCCH2CH2O
2 R = H
3 R = Bz
1
4
ii
Scheme 1. Reagents and conditions (yields): (i) (a) NaIO4, 1,4-dioxane/water, (b)
NaBH4, 1,4-dioxane/water [ABz: 86%, CAc: 71%, GiBu: 68%, U: 82%]; (ii) BzCl, 2 equiv
DBU (for U: 10 equiv pyridine), DCM, ꢁ70 °C [ABz: 73%, CAc: 64%, GiBu: 63%, U: 79%];
(iii) 2-cyanoethyl N,N-diisopropylphosphoramidochloridite, DIPEA, acetonitrile
[ABz: 71%, CAc: 44%, GiBu: 78%, U: 68%]. The following protected base derivatives
were applied in compounds 1–4: ABz = 6-N-benzoyladenin-9-yl, CAc = 4-N-acety-
The nearly identical CD spectra of the UNA-modified RNA du-
plexes ON3:RNA and ON4:RNA and the unmodified ON1:RNA du-
plex are in the 200–310 nm range (Fig. 2) and reveal that one or
two UNA monomers leave the bulk part of the overall helical
lcytosin-1-yl,
G
iBu = 2-N-isobutyrylguanin-9-yl,
U = uracil-1-yl,
DMT = 4,40-
dimethoxytrityl.

5422
N. Langkjꢀr et al. / Bioorg. Med. Chem. 17 (2009) 5420–5425
Table 1
Thermal stability of RNA:DNA and RNA:RNA duplexes containing one or two UNA monomers and effects of mismatchesa
Tm value (°C)
RNA strand
DNA complement
RNA complement
50-UGC ACU GUA UGU CUG UAC CAU (ON1)
50-UGC ACU GUA UGU CUG UAC CAU (ON2)
50-UGC ACU GUA UGU CUG UAC CAU (ON3)
50-UGC ACU GUA UGU CUG UAC CAU (ON4)
50-UGC ACU GUA UGU CUG UAC CAU (ON5)
50-ACU UGU GGC CAU UUA CGU CGC (ON6)
50-ACU UGU GGC CAU UUA CGU CGC (ON7)
50-ACU UGU GGC CAU UUA CGU CGC (ON8)
58 [ꢁ11, ꢁ9, ꢁ5]
50 [ꢁ4, ꢁ2, ꢁ3]
51 [ꢁ6, ꢁ7, ꢁ5]
41 [ꢁ1, 0, ꢁ1]
41 [ꢁ17, ꢁ13, ꢁ7]
61 [ꢁ6, ꢁ7, ꢁ6]
53 [ 0, ꢁ2, ꢁ1]
41 [ꢁ12, ꢁ10, ꢁ10]
71 [ꢁ10, ꢁ8, ꢁ6]
64 [ꢁ4, ꢁ2, ꢁ3]
66 [ꢁ8, ꢁ7, ꢁ6]
56 [ꢁ1, ꢁ1, ꢁ1]
47 [ꢁ12, ꢁ10, ꢁ9]
74 [ꢁ6, ꢁ6, ꢁ6]
66 [ꢁ1, ꢁ1, ꢁ1]
57 [ꢁ7, ꢁ7, ꢁ7]
a
Thermal denaturation temperatures (Tm values/°C) of unmodified or modified RNA duplexes measured as the maximum of the first derivative of the melting curve (A260 vs
M concentrations of the two complementary
Tm values for mismatches at position 11 (central G or A monomer) of the RNA strands ON1–ON5 are shown in
Tm values in brackets are shown in the following order: [G:A mismatch, G:G mismatch and G:T/U mismatch or A:A mismatch, A:C mismatch, and A:G
temperature) recorded in medium salt buffer (100 mM NaCl, 0.1 mM EDTA, 10 mM NaH2PO4, 5 mM Na2HPO4, pH 7.0), using 1.0
strands. Tm values are average of at least two measurements.
brackets; the
l
D
D
mismatch] and are calculated relative to the fully matched duplexes (G:C or A:T/U match). A = adenin-9-yl monomer, C = cytosin-1-yl monomer, G = guanine-9-yl monomer,
U = uracil-1-yl monomer; UNA monomers are bold letters.
geometry unperturbed. All the three CD spectra show the charac-
teristic features of an A-type helical environment with negative
bands of rather low intensities at ꢀ210 and ꢀ235 nm, and a posi-
tive band of strong intensity at ꢀ265 nm. The intensity of the
bands is only slightly less intense for UNA-modified RNA duplexes.
These spectra reveal that a UNA monomer structurally can be con-
sidered as an RNA mimic, which suggests applicability of UNA-
modified RNA ONs for biological and biotechnological applications.
One such application is UNA-modified siRNA for which highly po-
tent silencing with improved selectivity has been recently
reported.27
4. Experimental
4.1. General
Reactions under anhydrous conditions were carried out under
an atmosphere of nitrogen. After column chromatographic purifi-
cations, the fractions containing product were pooled and evapo-
rated to dryness under reduced pressure. After drying organic
phases over Na2SO4, filtration was performed. Solvents were of
HPLC grade, of which acetonitrile, DCM and pyridine were dried
over molecular sieves (4 Å). TLC was run on Merck Silica 60 F254
aluminum sheets. 1H NMR spectra were recorded at 300 MHz,
13C NMR spectra at 75.5 MHz and 31P NMR spectra at 121.5 MHz
(dH: DMSO-d6 2.50; dC: DMSO-d6 39.4). Chemical shifts are re-
ported in ppm relative to either tetramethylsilane or the deuter-
ated solvent as a internal standard for 1H NMR and 13C NMR, and
relative to 85% H3PO4 as an external standard for 31P NMR. Assign-
ments of NMR spectra, when given, are based on 2D NMR experi-
ments (the assignments of methylene protons/methylene carbons
may be interchanged). Coupling constants (J values) are given in
Hz. ESI-HRMS and MALDI-HRMS were recorded in positive ion
mode on an Ion Spec Fourier transform mass spectrometer.
3. Conclusion
Synthesis of UNA phosphoramidite derivatives suitable for
highly efficient incorporation of UNA monomers of the four natural
bases into RNA strands has been accomplished. UNA monomers
destabilize duplexes formed between UNA-modified RNA strands
and complementary RNA or DNA, and two UNA monomers can
be used to decrease or increase mismatch discrimination depend-
ing on their sites of incorporation. UNA has herein been introduced
as an RNA mimicking novel tool for engineering of nucleic acid du-
plex stability and base pairing specificity.
4.2. 50-O-(4,40-Dimethoxytrityl)-20,30-secouridine (2U)28
50-O-(4,40-Dimethoxytrityl)uridine (1U, 10.35 g, 18.94 mmol)
was at rt dissolved in a stirred mixture of dioxane (250 ml) and
water (50 ml). To this solution was added NaIO4 (4.47 g,
20.90 mmol) dissolved in water (50 ml). The resulting mixture
was stirred for 1 h during which time a white precipitate was
formed. Additional dioxane (200 ml) was added and the suspen-
sion was stirred for additional 15 min, whereupon the suspension
was filtered through a glass filter and the filter cake was washed
with dioxane (100 ml). The filtrates were combined, NaBH4
(797 mg, 21.1 mmol) was added, and the resulting mixture was
stirred for 30 min. The mixture was neutralized by the addition
of buffer (pyridine:AcOH 1:1, v/v, ꢀ10 ml). After evaporation of
the resulting mixture to a volume of approximately 150 ml, DCM
(100 ml) was added and the mixture was washed with sat. aq.
NaHCO3 (2 ꢂ 100 ml). The organic phase was separated and evap-
orated to dryness under reduced pressure. The crude product was
purified by column chromatography (40% acetone in petroleum
ether) affording the desired nucleoside 2U as a white foam
(8.53 g, 82%). Rf: 0.2 (10% MeOH in DCM). 1H NMR (DMSO-d6): d
11.34 (br s, NH), 7.62 (d, 1H, J = 8.0, H6), 7.34–7.15 (m, 9H, Ar),
6.85 (d, 4H, J = 8.0, Ar), 5.80 (t, 1H, J = 5.8, H10), 5.52 (d, 1H,
J = 8.0, H5), 5.12 (t, 1H, J = 5.9, 20OH), 4.74 (t, 1H, J = 5.4, 30OH),
3.72 (s, 6H, OCH3), 3.55–3.47 (m, 3H, H20/H40), 3.40 (t, 2H, J = 5.2,
8
6
4
2
0
-2
-4
220
240
260
280
300
320
340
Wavelength [nm]
Figure 2. CD spectra of unmodified (ON1:RNA, solid line) and UNA-modified RNA
duplexes (ON3:RNA, dashed line; ON4:RNA, dotted line).

N. Langkj
ꢀr et al. / Bioorg. Med. Chem. 17 (2009) 5420–5425
5423
H30), 3.01–2.90 (m, 2H, H50). 13C NMR (DMSO-d6): d 163.2, 157.9,
151.4, 144.8, 141.1 (C6), 135.4, 129.5, 127.7, 127.5, 126.5, 113.0,
101.6 (C5), 83.6 (C10), 79.3 (C20/C40), 63.5 (C50), 61.1 (C20/C40),
60.5 (C30), 54.9 (OCH3). ESI-MS (M+Na+): m/z 571.2; calcd:
571.2051.
of the resulting mixture to a volume of approximately 100 ml,
DCM (100 ml) was added and the mixture was washed with sat.
aq. NaHCO3 (2 ꢂ 100 ml). The organic phase was evaporated to
dryness under reduced pressure to give a residue which was puri-
fied by column chromatography (0–10% iPrOH in DCM) to give
nucleoside 2ABz as a white solid material (6.07 g, 86%). Rf: 0.22
(5% iPrOH in DCM). 1H NMR (DMSO-d6): d 11.23 (br s, 1H), 8.76
(s, 1H), 8.68 (s, 1H), 8.07 (d, 2H, J = 7.0, Ar), 7.69–7.50 (m, 3H,
Ar), 7.25–6.91 (m, 9H, Ar), 6.81–6.78 (m, 4H, Ar), 6.06 (t, 1H,
J = 6.1, H10), 5.29 (t, 1H, J = 5.8, 20OH), 4.84 (t, 1H, J = 5.4, 30OH),
4.19–4.02 (m, 2H, H20), 3.90–3.80 (m, 1H, H40), 3.69 (s, 6H,
OCH3), 3.49 (t, 2H, J = 5.3, H30), 2.93–2.74 (m, 2H, H50). 13C NMR
(DMSO-d6): d 165.6, 157.9, 152.8, 151.6, 150.2, 144.6, 143.1,
135.7, 135.4, 132.4, 129.4, 128.5, 128.4, 127.7, 127.5, 126.4,
125.2, 113.0, 84.5 (10C), 79.6 (40C), 63.6 (50C), 61.4 (20C), 60.9
(30C), 54.9 (OCH3). ESI-HRMS (M+Na+): m/z 698.2613; calcd:
698.2585.
4.3. 20-O-Benzoyl-50-O-(4,40-dimethoxytrityl)-20,30-secouridine
(3U)28
Nucleoside 2U (3.01 g, 5.50 mmol) was coevaporated with
anhydrous toluene (15 ml) and dried for 12 h in vacuo. The result-
ing residue was dissolved in anhydrous DCM (150 ml) along with
anhydrous pyridine (4.4 ml) and the mixture was cooled to
ꢁ78 °C. Benzoyl chloride (700
ll, 6.0 mmol) was added to the mix-
ture over 15 min and stirring was continued for 1 h at ꢁ78 °C. The
mixture was allowed to warm to rt and EtOH (5 ml) was added.
The resulting mixture was washed successively with sat. aq. NaH-
CO3 (3 ꢂ 100 ml) and brine (100 ml). The combined aqueous phase
was extracted with DCM (100 ml) and the organic phases were
combined and evaporated to dryness under reduced pressure.
The resulting residue was purified by column chromatography
(3.5% MeOH in DCM) affording the product 3U as a white foam
(3.44 g, 79%). Rf: 0.3 (5% MeOH in DCM). 1H NMR (DMSO-d6):
d11.43 (s, 1H, NH), 7.93–7.87 (m, 2H, Ar), 7.80 (d, 1H, J = 8.0, H6),
7.70–7.63 (m, 1H), 7.56–7.48 (m, 2H, Ar), 7.35–7.17 (m, 10H, Ar),
6.89–6.81 (m, 4H, Ar), 6.20 (t, 1H, J = 5.7, H10), 5.56 (d, 1H, J = 8.0,
H5), 4.83 (t, 1H, J = 5.2, OH-30), 4.71–4.45 (m, 2H), 3.73 (s, 6H,
OCH3), 3.70–3.62 (m, 1H, H40), 3.50–3.40 (m, 2H, H30), 3.11–2.96
(m, 2H, H50). 13C NMR (DMSO-d6): d 165.01, 163.08, 157.99,
151.10, 144.77, 140.61, 135.55, 135.45, 133.64, 129.55, 129.12,
129.02, 128.84, 127.74, 127.61, 126.59, 113.11, 102.16, 85.45,
80.94, 79.67, 63.47, 60.59, 54.98, 54.90. ESI-MS (M+Na+): m/z
675.2; calcd: 675.2313.
4.6. 6-N-Benzoyl-20-O-benzoyl-50-O-(4,40-dimethoxytrityl)-20,30-
secoadenosine (3ABz
)
Nucleoside 2ABz (2.01 g, 2.98 mmol) was coevaporated with
anhydrous MeCN (2 ꢂ 30 ml) and dried for 12 h in vacuo. The res-
idue was dissolved in anhydrous DCM (150 ml) along with anhy-
drous DBU (900 mg, 5.96 mmol) and the resulting mixture was
cooled to ꢁ78 °C. A 0.5 M solution of benzoyl chloride (6.56 ml,
3.28 mmol) was added over 15 min and the resulting mixture
was stirred for 1 h at ꢁ78 °C and was subsequently allowed to
reach rt, whereupon EtOH (5 ml) was added. The mixture was then
washed successively with sat. aq. NaHCO3 (3 ꢂ 150 ml) and brine
(150 ml) and the combined aqueous phase was back-extracted
with DCM (100 ml). The organic phases were combined and evap-
orated to dryness under reduced pressure. The resulting residue
was purified by column chromatography (0–100% EtOAc in petro-
leum ether) affording the product nucleoside 3ABz as a white foam
(1.69 g, 73%). Rf: 0.49 (EtOAc). 1H NMR (DMSO-d6): d 11.28 (s, 1H,
NH), 8.82 (s, 1H), 8.76 (s, 1H), 8.06 (d, 2H, J = 7.1, Ar), 7.83–7.78 (m,
2H), 7.55–7.40 (m, 6H, Ar), 7.25–6.89 (m, 10, Ar), 6.82–6.74 (m,
4H), 6.51 (t, 1H, J = 6.1, H10), 5.12–4.93 (m, 2H, H20), 4.90 (t,
J = 5.3, 30OH), 3.89 (br s, 1H, H40), 3.72 (s, 6H, OCH3), 3.54 (m, 2H,
H30), 2.81 (m, 2H, H50). 13C NMR (DMSO-d6): d 165.0, 157.9,
152.4, 150.5, 144.6, 142.9, 135.6, 133.6, 132.4, 129.0, 128.8,
128.4, 127.5, 125.2, 113.0, 85.1, 81.5 (C10), 79.9 (C40), 63.8 (C20),
63.5, 60.8 (C50), 54.9 (OCH3). ESI-HRMS (M+Na+): m/z 802.2848;
calcd: 802.2847.
4.4. 20-O-Benzoyl-30-O-(2-cyanoethoxy(diisopropylamino)phos-
phino)-50-O-(4,40-dimethoxytrityl)-20,30-secouridine (4U)28
Nucleoside 3U (679 mg, 1.04 mmol) was coevaporated with
DCE (3 ꢂ 6 ml) and dried for 12 h in vacuo. The residue was dis-
solved at rt in 20% DIPEA in MeCN (6.5 ml) and 2-cyanoethyl-
N,N-diisopropylchlorophosphoramidite
[P(Cl)(OCH2CH2CN)(-
N(iPr)2)] (0.66 ml, 3.02 mmol) was added and stirring was contin-
ued for 40 min. The mixture was then poured into DCE (10 ml) and
washed with sat. aq. NaHCO3 (10 ml), and the separated aqueous
phase was back-extracted with DCE (10 ml). The organic phases
were pooled and evaporated to dryness to afford at residue which
was purified by column chromatography (0–20% EtOAc in toluene)
to give nucleoside 4U as a white solid material (600 mg, 68%). Rf:
0.6 (50% EtOAc in toluene). 31P NMR (MeCN): d 147.8. ESI-MS
(M+Na+): m/z 875.3; calcd: 875.3391.
4.7. 30-O-(2-Cyanoethoxy(diisopropylamino)phosphino)-6-N,20-
O-dibenzoyl-50-O-(4,40-dimethoxytrityl)-20,30-secoadenosine
(4ABz
)
Nucleoside 3ABz (1.69 g, 2.17 mmol) was coevaporated with
anhydrous MeCN (2 ꢂ 20 ml) and was dried for 12 h in vacuo.
The residue was dissolved in 20% DIPEA in MeCN (40 ml) and 2-
4.5. 6-N-Benzoyl-50-O-(4,40-dimethoxytrityl)-20,30-secoadenosine
(2ABz
)
cyanoethyl-N,N-diisopropylchlorophosphoramidite
4.48 mmol) was added at rt under stirring. After 40 min, additional
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.2 ml,
(1.0 ml,
6-N-Benzoyl-50-O-(4,40-dimethoxytrityl)adenosine
(1ABz
;
7.02 g, 10.42 mmol) was at rt dissolved in dioxane (150 ml) and
water (25 ml). To this solution was added NaIO4 (2.73 g,
11.77 mmol) dissolved in water (25 ml). The resulting mixture
was stirred for 1 h during which time a white precipitate was
formed. Additional dioxane (100 ml) was added and the suspen-
sion was stirred for additional 15 min, whereupon the suspension
was filtered through a glass filter and the filter cake was washed
with dioxane (50 ml). The filtrates were combined, NaBH4
(435 mg, 11.5 mmol) was added and the resulting mixture was
stirred for 30 min. The mixture was then neutralized by the addi-
tion of buffer (pyridine:AcOH 1:1, v/v, ꢀ10 ml). After evaporation
0.86 mmol) was added and stirring was continued for 3 h, where-
upon EtOH (5 ml) was added. The resulting mixture was washed
with sat. aq. NaHCO3 (3 ꢂ 50 ml) and the aqueous phases were
combined and extracted with DCM (50 mL). The organic phases
were pooled and evaporated to dryness under reduced pressure
to give a residue which was purified by column chromatography
(0–100% EtOAc in petroleum ether) to afford the desired nucleo-
side 4ABz as a white foam (1.52 g, 71%). Rf: 0.75 (5% MeOH in
DCM). 31P NMR (MeCN):
d
148.8. ESI-HRMS (M+Na+): m/z
1002.3885; calcd: 1002.3926.

5424
N. Langkjꢀr et al. / Bioorg. Med. Chem. 17 (2009) 5420–5425
4.8. 4-N-Acetyl-50-O-(4,40-dimethoxytrityl)-20,30-secocytidine
(2CAc
PEA in MeCN (20 ml) and the mixture was stirred at rt. 2-Cyano-
ethyl-N,N-diisopropylchlorophosphoramidite (0.8 ml, 3.6 mmol)
was added to the mixture and stirring was continued for 40 min.
Additional 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite
(0.4 ml, 1.8 mmol) was added and stirring was continued for 3 h.
EtOH (5 ml) was added and the resulting mixture was washed with
sat. aq. NaHCO3 (3 ꢂ 50 ml) and the aqueous phase was extracted
with DCM (50 ml). The organic phases were pooled and evaporated.
The residue was purified by column chromatography (0–100%
EtOAc in petroleum ether) to afford nucleoside 4CAc as a white foam
(940 mg, 44%). Rf: 0.42 (5% MeOH in DCM). 31P NMR (MeCN): d
148.8. ESI-HRMS (M+Na+): m/z 916.3622; calcd: 916.3657.
)
4-N-Acetylcytidine (11.75 g, 41.18 mmol) was coevaporated
with anhydrous pyridine (50 ml) and then dissolved in anhydrous
pyridine (160 ml). 4,40-Dimethoxytrityl chloride (DMT-Cl, 16.76 g,
49.42 mmol) was added as a solid material and the resulting mix-
ture was stirred for 2 h at rt. The mixture was then washed with
sat. aq. NaHCO3 (3 ꢂ 50 ml) and the organic phase was evaporated
to dryness under reduced pressure. This residue was dissolved in a
mixture of dioxane (500 ml) and water (100 ml), and NaIO4
(10.62 g, 49.5 mmol) dissolved in water (100 ml) was added. The
mixture was stirred for 1 h during which time a white precipitate
was formed. Additional dioxane (400 ml) was added and stirring
was continued for 15 min. The precipitate was filtered off and
washed with dioxane (200 ml). The filtrates were combined,
NaBH4 (1720 mg, 45.5 mmol) was added, and stirring was contin-
ued for 30 min. To the mixture was added a buffer (AcOH:pyridine,
1:1, 10 ml) and the resulting mixture was evaporated to a volume
of approximately 300 ml. EtOAc (150 ml) was added and washing
was performed with sat. aq. NaHCO3 (3 ꢂ 200 ml). The organic
phase was evaporated to dryness under reduced pressure and the
residue was purified by column chromatography (0–10% MeOH
in EtOAc) to afford nucleoside 2CAc as a white solid material
(17.24 g, 71%). Rf: 0.19 (5% MeOH in CHCl3). 1H NMR (DMSO-d6):
d 10.94 (s, 1H, NH), 8.08 (d, 1H, J = 7.4), 7.31–7.12 (m, 12H),
6.95–6.75 (m, 4H, Ar), 5.96 (t, 1H, J = 4.8, H10), 5.13 (t, 1H, J = 5.9,
20OH), 4.74 (t, 1H, J = 5.5, 30OH), 3.73 (s, 6H, OCH3), 3.63 (m, 3H,
H20/H40), 3.43 (m, 2, H30), 3.00 (m, 2H, H50), 2.13 (s, 3H, CH3). 13C
NMR (DMSO-d6): d 170.9, 162.3, 158.0, 155.4, 146.1, 144.6, 135.6,
129.6, 127.7, 126.6, 113.1, 95.4, 85.5, 84.7 (C10), 79.4, 63.8 (C50),
61.7, 60.5 (C30), 55.0 (OCH3), 24.3 (CH3). ESI-HRMS (M+Na+): m/z
612.2298; calcd: 612.2316.
4.11. 50-O-(4,40-Dimethoxytrityl)-2-N-isobutyryl-20,30-
secoguanosine (2GiBu
)
2-N-Isobutyrylguanosine (11.68 g, 17.8 mmol) was coevaporat-
ed with anhydrous pyridine (50 ml). The resulting residue was dis-
solved in anhydrous pyridine (100 ml) and DMT-Cl (7.26 g,
21.46 mmol) was added as a solid material and the reaction mix-
ture was stirred for 1 h at rt. DMAP (50 mg, 0.40 mmol) was added
and the resulting mixture was stirred for additional 12 h. The reac-
tion mixture was then washed with sat. aq. NaHCO3 (3 ꢂ 50 ml)
and the organic phase was evaporated to yield a white foam. This
residue was dissolved in dioxane (250 ml) and water (50 ml).
NaIO4 (4.57 g, 21.3 mmol) was dissolved in water (50 ml) and
was added to the dissolved nucleoside. The mixture was stirred
for 1 h during which time a white precipitate was formed. Addi-
tional dioxane (200 ml) was added and stirring was continued for
15 min. The precipitate was filtered off and washed with dioxane
(100 ml). The filtrates were collected and NaBH4 (748 mg,
19.77 mmol) was added and the resulting mixture was stirred for
30 min at rt. The mixture was neutralized by the addition of buffer
(10 ml, 1:1–AcOH:pyridine). The volume of the resulting mixture
was reduced to 150 ml and EtOAc (150 ml) was added. The organic
phase was washed with sat. aq. NaHCO3 (3 ꢂ 100 ml) and evapo-
rated. The residue was purified by column chromatography using
a gradient of 0–10% MeOH in DCM to yield the nucleoside 2GiBu
as a white material (8.02 g, 68%). Rf: 0.24 (7% MeOH in DCM). 1H
NMR (DMSO-d6): d 12.10 (s, 1H, NH), 11.71 (s, 1H, NH), 8.17 (s,
1H, guanidine H8), 7.25–6.94 (m, 10H, Ar), 6.82–6.74 (m, 4H, ar),
5.72 (t, 1H, J = 6.2, H10), 5.19 (t, 1H, J = 5.8, 20OH), 4.79 (t, 1H,
J = 5.2, 30OH), 3.99 (t, 2H, J = 6.1, H20), 3.83–3.67 (m, 8H, H40/
OCH3), 3.48–3.40 (m, 2H, H30), 2.93–2.73 (m, 2H, H50/tertiary i-
Pr), 1.12 (dd, 6H, J = 6.8, 2.6., 2 ꢂ CH3). 13C NMR (DMSO-d6): d
180.2, 157.9, 154.9, 147.8, 144.7, 135.4, 129.3 (Ar), 127.5 (Ar),
127.4 (Ar), 126.4 (Ar), 120.4, 113.0 (Ar), 85.2, 85.1 (C10), 79.9
(C40), 63.5 (C50), 61.7 (C20), 61.1 (C30), 54.9 (–OCH3), 34.7 (tertiary
i-Pr), 18.9 (i-Pr), 18.8 (i-Pr). MALDI-HRMS (M+Na+): m/z
680.2679; calcd: 680.2691.
4.9. 4-N-Acetyl-20-O-benzoyl-50-O-(4,40-dimethoxytrityl)-20,30-
secocytidine (3CAc
)
Nucleoside 2CAc (3.03 g, 5.14 mmol) was coevaporated with
anhydrous toluene (2 ꢂ 30 ml) and was dried for 12 h in vacuo.
The resulting residue was dissolved in anhydrous DCM (150 ml)
along with anhydrous DBU (1.5 ml, 10.3 mmol) and the resulting
mixture was cooled to ꢁ78 °C. Benzoyl chloride (656
ll,
5.65 mmol) was slowly added over 5 min and stirring was contin-
ued for 1 h at ꢁ78 °C. The mixture was then allowed to warm to
rt, whereupon EtOH (4 ml) was added. The mixture was washed
with sat. aq. NaHCO3 (2 ꢂ 150 ml) and the organic phase was evap-
orated to dryness under reduced pressure. The resulting residue
was purified by column chromatography (0–5% MeOH in CHCl3)
affording product nucleoside 3CAc as a white foam (2.08 g, 64%).
Rf: 0.24 (5% MeOH in CHCl3). 1H NMR (DMSO-d6): d 10.98 (s, 1H,
NH), 8.24 (d, J = 7.4, 1H), 7.93–7.85 (m, 2H), 7.69–7.60 (m, 1H),
7.54–7.44 (m, 2H), 7.33–7.13 (m, 12H), 6.89–6.79 (m, 4H), 6.34 (t,
J = 5.0, 1H, H10), 4.83 (t, J = 5.4, 1H, 30OH), 4.67–4.48 (m, 2H, H20),
3.72 (s, 6H, OCH3), 3.70–3.62 (m, 1H, H40), 3.55–3.39 (m, 2H, H30),
3.11–2.93 (m, 2H, H50), 2.12 (s, 3H, CH3). 13C NMR (DMSO-d6): d
171.0, 165.0, 162.5, 157.1, 145.5, 144.6, 135.48, .133.6, 129.6,
128.8, 127.7, 127.6, 126.6, 113.1, 95.8, 85.6, 82.0 (C10), 79.6 (C40),
79.2 63.9 (C50), 63.7 (C20), 60.5 (C30), 54.9 (OCH3), 24.3 (CH3). ESI-
HRMS (M+Na+): m/z 716.2589; calcd: 716.2759.
4.12. 20-O-Benzoyl-50-O-(4,40-dimethoxytrityl)-2-N-isobutyryl-
20,30-secoguanosine (3GiBu
)
Nucleoside 2GiBu (2.01 g, 3.05 mmol) was suspended in anhy-
drous toluene (2 ꢂ 30 ml) and evaporated. The resulting residue
was dried for 12 h in vacuo and then dissolved in anhydrous DCM
(100 ml) along with anhydrous DBU (0.9 ml, 6.1 mmol). The result-
ing mixture was cooled to ꢁ78 °C. Benzoyl chloride (390
ll,
3.36 mmol) was added over 5 min to the reaction mixture and stir-
ring was continued for 1 h at ꢁ78 °C. The reaction mixture was then
allowed to reach rt, whereupon EtOH (4 ml) was added. The result-
ing mixture was washed with sat. aq. NaHCO3 (2 ꢂ 100 ml), and the
organic phases were combined and evaporated. The resulting resi-
due was purified by column chromatography (0–5% MeOH in
CHCl3) affording nucleoside 3GiBu as a white foam (1.49 g, 63%).
4.10. 4-N-Acetyl-20-O-benzoyl-30-O-(2-cyanoethoxy(diisopro-
pylamino)phosphino)-50-O-(4,40-dimethoxytrityl)-20,30-
secocytidine (4CAc
)
Nucleoside 3CAc (1.49 g, 2.15 mmol) was coevaporated with
anhydrous MeCN (2 ꢂ 20 ml). The residue was dissolved in 20% DI-

N. Langkj
ꢀr et al. / Bioorg. Med. Chem. 17 (2009) 5420–5425
5425
Rf: 0.47 (7% MeOH in DCM). 1H NMR (DMSO-d6): d 12.10 (s, 1H, NH),
11.72 (s, 1H, NH), 8.32 (s, 1H, guanidine H8), 7.85–7.79 (m, 2H, Ar),
7.65–7.63 (m, 1H, Ar), 7.51–7.45 (m, 2H, Ar), 7.26–6.97 (m, 11H, Ar),
6.81–6.77 (m, 4H, Ar), 6.18 (t, 1H, J = 6.2, H10), 5.04–4.82 (m, 3H,
H20/300OH), 3.82 (m, 1H, H40), 3.72 (s, 6H, 2 ꢂ OCH3), 3.49 (t, 2H,
J = 5.1, H30), 3.03–2.74 (m, 3H, H50/quaternary i-Pr), 1.15–1.04 (m,
6H, 2 ꢂ CH3). 13C NMR (DMSO-d6): d 180.1, 164.9, 157.8, 154.8,
148.6, 147.9, 144.6, 138.4, 135.5, 135.3, 133.6, 129.3 (Ar), 129.0
(ar), 128.8 (ar), 128.7 (ar), 127.6 (ar), 127.4 (ar), 126.3 (ar), 120.6,
112.9 (ar), 85.1, 82.0 (C10), 80.1 (C40), 63.7, 63.3 (C50), 61.0 (C30),
54.8 (OCH3), 34.6 (quaternary i-Pr), 18.8 (CH3), 18.6 (CH3). ESI-
HRMS (M+Na+): m/z 784.294; 3 calcd: 784.2953.
gonucleotide single strand concentrations were calculated based
on the absorbance values measured at rt. The samples containing
2 lM solutions of each duplex were prepared in a medium salt buf-
fer (see caption below Table 1 for details), annealed for two min-
utes at 90 °C and slowly cooled to room temperature before the
experiment. The buffer spectrum was subtracted from the duplex
spectra. The spectra were smoothed using Savitzky–Golay filter.
Acknowledgments
The Nucleic Acid Center is a research center of excellence
funded by the Danish National Research Foundation for studies
on nucleic acid chemical biology. We gratefully acknowledge
financial support by The Danish National Research Foundation
and the Sixth Research Framework Programme of the European
Union, Project RIGHT (LSHB-CT-2004 005276).
4.13. 20-O-Benzoyl-30-O-(2-cyanoethoxy(diisopropylamino)-
phosphino)-50-O-(4,40-dimethoxytrityl)-2-N-isobutyryl-20,30-
secoguanosine (4GiBu
)
Nucleoside 3GiBu (1.45 g, 1.9 mmol) was coevaporated with
anhydrous MeCN (2 ꢂ 20 ml). The residue was dissolved in 20% DI-
PEA in MeCN (20 ml) and the resulting mixture was stirred. 2-Cya-
noethyl-N,N-diisopropylchlorophosphoramidite (0.65 ml, 2.91
mmol) was added to the reaction mixture and stirring was contin-
ued for 40 min. EtOH (5 ml) was added and the resulting mixture
was washed with sat. aq. NaHCO3 (3 ꢂ 50 ml), and the aqueous
phase was extracted with DCM (50 ml). The organic phases were
pooled and evaporated. The residue was precipitated by adding a
solution of the crude in EtOAc to vigorously stirring petroleum
ether to furnish amidite 4GiBu as a white material (607 mg, 33%).
Rf: 0.3 (33% Acetone in toluene). 31P NMR (MeCN): d 148.6, 148.7.
ESI-HRMS (M+Na+): m/z 984.4028; calcd: 984.4031.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.06.045.
References and notes
1. Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25, 4429.
2. Wengel, J. Acc. Chem. Res. 1999, 32, 301.
3. Micklefield, J. Curr. Med. Chem. 2001, 8, 1157.
4. Kurreck, J. Eur. J. Biochem. 2003, 270, 1628.
5. Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.; Meldgaard,
M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54, 3607.
6. Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T.
Tetrahedron Lett. 1998, 38, 8735.
7. Vester, B.; Wengel, J. Biochemistry 2004, 43, 13233.
4.14. Synthesis of UNA-modified RNA oligonucleotides29
8. Jepsen, J. S.; Wengel, J. Curr. Opin. Drug Discovery 2004, 7, 188.
9. Ørum, H.; Wengel, J. Curr. Opin. Mol. Ther. 2001, 3, 239.
10. Ørum, H.; Jacobsen, M. H.; Koch, T.; Vuust, J.; Borre, M. B. Clin. Chem. 1999, 45,
1898.
11. Petersen, M.; Bondensgaard, K.; Wengel, J.; Jacobsen, J. P. J. Am. Chem. Soc. 2002,
124, 5974.
The UNA-modified and unmodified RNA oligonucleotides (Table
1) were synthesized on an automated nucleic acid synthesizer
using the phosphoramidite approach. The syntheses were per-
12. Nielsen, K. E.; Rasmussen, J.; Kumar, R.; Wengel, J.; Jacobsen, J. P.; Petersen, M.
Bioconjugate Chem. 2004, 15, 449.
formed in 1.0 lmol scale. Standard RNA synthesis conditions of
13. Nielsen, P.; Dreiøe, L. H.; Wengel, J. Bioorg. Med. Chem. 1995, 3, 19.
14. Mangos, M. M.; Min, K.-L.; Viazovkina, E.; Galarneau, A.; Elzagheid, M. I.;
Parniak, M. A.; Damha, M. J. J. Am. Chem. Soc. 2003, 125, 654.
15. Itkes, A. V.; Karpeisky, M. Y.; Kartasheva, O. N.; Mikhailov, S. N.; Moiseyev, G.
P.; Pfleiderer, W.; Charubala, R.; Yakovlev, G. I. FEBS Lett. 1988, 236, 325.
16. Mikhailov, S. N.; Pfleiderer, W. Tetrahedron Lett. 1985, 26, 2059.
17. Thermal denaturation data for UNA-modified duplexes are throughout the text
discussed relative to data obtained for the corresponding unmodified duplexes
involving ON1 or ON6.
18. Supplementary electronic information available: Procedures used for
thermodynamic analysis and table with thermodynamic parameters.
19. Kaur, H.; Wengel, J.; Maiti, S. Biochemistry 2008, 47, 1218.
20. McTigue, P. M.; Peterson, R. J.; Kahn, J. D. Biochemistry 2004, 43, 5388.
21. Seela, F.; Debelak, H. Nucleic Acids Res. 2000, 28, 3224.
22. Berger, M.; Wu, Y. Q.; Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.; Romesberg, F.
E. Nucleic Acids Res. 2000, 28, 2911.
the synthesizer were used for the incorporation of RNA and UNA
monomers using DCI as an activator, and the stepwise coupling
yield of unmodified RNA as well as UNA monomers were >99%
based on the absorbance of the dimethoxytrityl cation released
after each coupling step. After detritylation, cleavage from the solid
support and deacylation was carried out by using 30% aqueous
ammonia solution. Cleaving of protecting groups was achieved
by the addition of methylamine to the above solution to furnish
a 1:1 mixture (2 h, 65 °C). Analysis by ion-exchange HPLC verified
the purity of all oligonucleotides to be >80%, whereas their compo-
sition was verified by MALDI-TOF mass spectrometry.
4.15. Thermal denaturation experiments
23. Babu, B. R.; Prasad, A. K.; Trikha, S.; Thorup, N.; Parmar, V. S.; Wengel, J. J. Chem.
Soc., Perkin Trans. 1 2002, 2509.
24. Van Aerschot, A.; Rozenski, J.; Loakes, D.; Pillet, N.; Schepers, G.; Herdewijn, P.
Nucleic Acids Res. 1995, 23, 4363.
25. Loakes, D. Nucleic Acids Res. 2001, 29, 2437.
26. We have systematically reduced the distance of the two UNA monomers to the
central position from five to zero RNA bases and found that the ‘distal UNA
design’ is the optimal with respect to mismatch discrimination.
UV-based thermal denaturation experiments were performed
on a Perkin–Elmer Lambda 35 UV/vis spectrometer equipped with
PTP 6 (Peltier Temperature Programmer) in a medium salt buffer
(see caption below Table 1 for details) using 1.0 lM concentration
of each strand. The two strands were thoroughly mixed, heated to
90 °C and subsequently cooled to the starting temperature of the
experiment (10 °C). Thermal denaturation temperatures (Tm val-
ues, °C) were determined as the maximum of the first derivative
of the thermal denaturation curve (A260 vs temperature; reported
Tm values are an average of two measurements within 1.0 °C).
27. J.B. Bramsen, M.B. Laursen, A.F. Nielsen, T.B. Hansen, C. Bus, N. Langkjꢀr, B.R.
Babu, T. Højland, M. Abramov, A. Van Aerschot, D. Odadzic, R. Smicius, J. Haas,
C. Andree, J. Barman, M. Wenska, P. Srivastava, C. Zhou, D. Honcharenko, S.
Hess, E. Müller, G.V. Bobkov, S.N. Mikhailov, E. Fava, T.F. Meyer, J.
Chattopadhyaya, M. Zerial, J.W. Engels, P. Herdewijn, J. Wengel, J. Kjems,
Nucleic Acids Res. 2009. doi:10.1093/nar/gkp106.
28. Pandolfi, D.; Rauzi, F.; Capobianco, M. L. Nucleosides, Nucleotides Nucleic Acids
1999, 18, 2051.
29. UNA phosphoramidite monomers and UNA-modified oligonucletides are
commercially available from RiboTask ApS.
4.16. Circular dichroism spectra
CD spectra were measured in a 5 mm cuvette on a Jasco J-600A
spectropolarimeter at 20 °C, in 200–350 nm wavelength range. Oli-